Nucleic acid molecule encoding human GM-CSF antigen binding proteins

ABSTRACT

Antigen binding proteins that bind to human GM-CSF protein are provided. Nucleic acids encoding the antigen binding protein, vectors, and cells encoding the same are also provided. The antigen binding proteins can inhibit binding of GM-CSF to GM-CSFR, inhibit GM-CSF-induced proliferation and signaling of myeloid lineage cell lines and inhibit GM-CSF-induced activation of human monocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/675,013, filed Jan. 18, 2011, which is a national stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US2008/010888,having an international filing date of Sep. 18, 2008; which claims thebenefit of U.S. provisional application Ser. No. 61/087,551, filed Aug.8, 2008, and U.S. provisional application Ser. No. 60/994,343, filedSep. 18, 2007, the disclosures of which are relied upon and incorporatedby reference herein.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-1292-US-DIV_Sequence_Listing.txt., created Aug. 15, 2012, which is 162KB in size. The information in the electronic format of the SequenceListing is incorporate herein by reference in its entirety.

BACKGROUND

Granulocyte macrophage colony stimulating factor (GM-CSF; CSF2) is awell-studied protein which has long been appreciated for itshematopoietic properties (i.e. stimulation of proliferation anddifferentiation of progenitor cells and proliferation of mature cells ofthe myeloid lineage) (reviewed in Blood 77:1131, 1991; Rev Infect Dis12: 41, 1990; Med. Oncol. 13:141, 1996). GM-CSF is constitutivelyproduced by lung epithelial cells and the Paneth cells of the intestine(BBRC 312:897, 2003), but a wide variety of cells express GM-CSF uponactivation with predominant expression from T cells,macrophages/monocytes, fibroblasts and endothelial cells (J Infect Dis172:1573, 1995; J Infect Dis 185:1490, 2002; J Allergy Cin Immunol112:653, 2003). The GM-CSF receptor (GM-CSFR; CSFR2) consists of aheterologous complex of two proteins; a high affinity alpha polypeptidewhich is specific for GM-CSF, and a low affinity common beta polypeptidewhich is shared by GM-CSF, IL-3 and IL-5 (reviewed in J Allergy CinImmunol 112:653, 2003; Cytokine and Growth Factor Reviews 12:19, 2001).GM-CSFR is expressed on all cells of the myeloid lineage.

GM-CSF augments the activity of the innate immune system by mediatingsignals that cause or effect differentiation, survival, proliferationand activation of myeloid lineage cells including macrophages/monocytes,dendritic cells (DCs), neutrophils and eosinophils (reviewed in: J Immun143:1198, 1989; Rev Infect Dis 12:41, 1990; Blood 77:1131, 1991; Trendsin Immun. 23:403, 2002; Growth Factors 22:225, 2004). GM-CSF is animportant factor for in vitro generation of monocyte-derived DCs andtype 1 macrophages (PNAS 101:4560, 2004), and has been shown to inducedifferentiation and activation of DCs in vivo (Blood 95:2337, 2000).Human monocyte-derived macrophages generated in the presence of GM-CSF(Type 1 macrophages) produce high levels of proinflammatory cytokinessuch as IL-23, but not IL-12, whereas Type 2 macrophages generated inthe presence of M-CSF (CSF1) produce anti-inflammatory cytokines such asIL-10, but not IL-23 (PNAS 101: 4560, 2004).

Human monocytes or macrophages stimulated with GM-CSF have increasedfunction including cytotoxicity, production of other proinflammatorycytokines TNFα and IL-6) and phagocytosis. Based on these effects, mucheffort has recently been applied to developing GM-CSF as a potentadjuvant for use in infectious disease or with administration ofvaccines (reviewed in Eur J Clin Microbiol Infect Dis. 13::S47, 1994;Curr Opin Hematol. 7:168, 2000). Indeed, administration of rhGM-CSF insome clinical settings dramatically improves outcome and clearance offungal infection (Eur J Clin Microbiol Infect Dis 13: S18, 1994; J MedMicrobiol 47: 1998).

Microglia are the resident macrophages of the CNS and data from in vitrostudies indicates that GM-CSF is a key cytokine which enhances survival,activation, proliferation and even differentiation of both fetal andadult microglial cells (Glia 12:309, 1994; J Immunol Methods 300:32,2005). In addition, there are several reports from mouse MS modelstudies which provide evidence for a critical role of APCs (microglia orDCs) in the perivascular space of the CNS for disease initiation andpersistence (Nat. Med. 11:146-2005; Nat. Med. 11:328, 2005; Nat. Med.11:335, 2005). GM-CSF stimulation of microglia upregulates MHCII andenhances antigen presentation

It is only recently that GM-CSF's role as a proinflammatory cytokine indisease, and dispensability as a hematopoietic growth factor, has beenestablished (reviewed in Trends in Immun. 23: 403, 2002; Growth Factors22: 225, 2004) and its role in causing or enhancinginflammatory/autoimmune disease.

Elevated levels of GM-CSF have been observed at local sites ofinflammation in multiple sclerosis (MS), rheumatoid arthritis (RA),asthma, psoriasis, atopic dermatitis and sarcoidosis. Elevation ofGM-CSF is not typically observed in the serum, thus determining diseaseassociation requires analysis of the target tissues. In MS, two clinicalstudies were performed in which levels of GM-CSF protein were measuredby ELISA in cerebrospinal fluid (CSF) and serum from Relapsing-Remitting(RR)MS patients with active disease (new symptoms or worsening ofexisting symptoms within 2 weeks of tissue collection) and compared witheither RRMS patients with stable disease (no episodes for prior 6months) or other neurological disease (OND) controls (Eur Neurol 33:152,1993; Immunopharmacol. Immunotoxicol. 20:373, 1998). Importantly, theOND controls did not include Alzheimer's Disease or vascular dementiapatients, as highly increased levels of GM-CSF were reported in the CSFand sera of such patients (Acta Neurol Scand 103:166, 2001).

GM-CSF levels were in the low pg range, but were significantly higher inRRMS active disease CSF compared to stable disease CSF, and in MS activedisease CSF compared to OND CSF. In addition, there were higher levelsof TNF-alpha in CSF of active versus stable disease, and higher levelsof both TGF-beta and IL-10 in CSF of stable versus active disease. Thestudies included very careful inclusion criteria with respect to ongoingtreatment of patients and clinical definition of active versus stabledisease, as well as synchronicity of sample collection. Interestingly,there were no significant differences in GM-CSF levels in serum betweenany of the groups. In addition, one study observed selectiveimmunohistochemical detection of GM-CSF in astrocytes of MS lesions andnot in control CNS white matter (n=3 MS donors, Glia 12:309, 1994).Finally, activated T cells and monocytes/macrophages are capable ofproducing large amounts of GM-CSF upon activation during an inflammatoryresponse. There is ample evidence for the presence of both of these celltypes in MS lesions (Ann Neurol. 47:707, 2000), and for T cells in CSF(reviewed in Curr. Neurol. Neurosci. Rep. 1: 257, 2001).

In addition to association of GM-CSF expression with MS, there is anabundance of disease association data for other inflammatory/autoimmunediseases and even some evidence for disease exacerbation withadministration of exogenous GM-CSF. In RA, elevated levels of GM-CSFhave been detected in synovial fluid (SF) of patients with RA orPsoriatic arthritis (PsA) compared to OA (bioassay, Clin. Exp. Immunol.72:67, 1988) and compared to non-RA controls (bioassay, Rheumatol Int.14:177, 1995). In addition, there is a strong correlation between thepresence of CD68+ macrophages in joints with disease severity in RApatients (Ann Rheum Dis 64:834, 2005). Finally, it has been reportedthat GM-CSF treatment of RA patients with Felty's syndrome (neutropenia)can exacerbate disease (Blood 74:2769, 1989).

In asthma, GM-CSF has been found to be elevated in bronchial biopsiesfrom asthmatic patients by immunohistochemistry and a correlation wasobserved between decrease in GM-CSF levels and increase in FEV1following steroid treatment (Chest 105:687, 1994; Am Rev Respir Dis147:1557, 1993). GM-CSF was also reported to be elevated in the sputumof intermittent, mild asthma patients (Ann Allergy Asthma Immunol86:304, 2001). Data to support antagonism of GM-CSF includes a study inwhich the eosinophil promoting activity from BALF of symptomaticpatients was attenuated by anti-GM-CSF mAb (in vitro, Eur. Respir. J.12:872, 1998).

In psoriasis, GM-CSF expression was detected in psoriatic skin but notcontrol skin samples (Arch Dermatol Res. 287:158, 1995; Clin ExpDermatol. 19:383, 1994; Dermatologica. 181:16, 1990). It has also beenreported that GM-CSF treatment of psoriasis can exacerbate disease (BrJ. Dermatol. 128:468, 1993).

In atopic dermatitis (AD), a significantly greater number of GM-CSF mRNAexpressing cells were detected by in situ hybridization in biopsies oflesions of chronic AD than in acute AD or nonlesion skin (p<0.05; J ClinInvest. 95:211, 1995). In a second study, higher levels of GM-CSF weredetected by immunohistochemistry of lesional AD skin (both epidermal anddermal compartments) and keratinocyte cultures established fromuninvolved skin of AD patients exhibited increased spontaneous andPMA-stimulated production of GM-CSF compared with keratinocytes fromnonatopic controls (J Clin Invest. 99:3009, 1997).

Mice deficient in GM-CSF (Science 264:713, 1994; PNAS 91:5592, 1994) andGM-CSFRc (Immunity 2:211, 1995; PNAS 92:9565, 1995) were generated bymultiple groups. The mice had no overt differences in steady statelevels of hematopoiesis, but did have histological evidence of alveolarproteinosis, were more susceptible to infections, and exhibited a modestdelay in IgG production and diminished antigen-specific T cell responsesafter KLH immunization (PNAS 94:12557, 1997). GM-CSF−/− mice areresistant to MOG35-55-induced EAE (J Exp Med. 194:873, 2001),collagen-induced arthritis (CIA; JI 161:3639, 1998) andmBSA/IL-1-induced arthritis (Arthritis Rheum 44:111, 2001). In contrast,GM-CSF transgenic (Tg) mice have been generated in a number of labs andare associated with the development of inflammatory/autoimmune disease(Cell 51:675, 1987; JI 166:2090, 2001; J Clin Invest 97:1102, 1996; JAllergy Clin Immunol 111:1076, 2003; Lab Invest 77:615, 1997).

Thus there is a need in the art for GM-CSF inhibitors.

SUMMARY

Antigen-binding proteins that bind GM-CSF, in particular human GM-CSF,are provided. The human GM-CSF antigen-binding proteins can inhibit,interfere with, or modulate at least one of the biological responsesrelated to GM-CSF, and, as such, are useful for ameliorating the effectsof GM-CSF-related diseases or disorders. Binding of certainantigen-binding proteins to GM-CSF can, therefore, inhibit, interferewith, or block GM-CSF signaling, reduce monocyte migration into tumors,and reduce the accumulation of tumor-associated macrophages (TAMs).

Also provided are expression systems, including cell lines, for theproduction of GM-CSF antigen binding proteins and methods for diagnosingand treating diseases related to human GM-CSF.

Some of the isolated antigen-binding proteins that are provided thatcomprise (A) one or more heavy chain complementary determining regions(CDRHs) selected from the group consisting of: (i) a CDRH1 selected fromthe group consisting of SEQ ID NO: 10, 22, 70 94 and 142; (ii) a CDRH2selected from the group consisting of SEQ ID NO: 11, 23, 28, 35, 47, 59,71, 95, 106, 119 and 143; (iii) a CDRH3 selected from the groupconsisting of SEQ ID NO: 12, 24, 36, 48, 60, 72, 83, 96, 108, 120, 132,and 144; and (iv) a CDRH of (i), (ii) and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more thanfour amino acids; (B) one or more light chain complementary determiningregions (CDRLs) selected from the group consisting of: (i) a CDRL1selected from the group consisting of SEQ ID NO: 4, 16, 30, 40, 52, 64,88, 100, 107, 112, 118, 124, 125 and 136; (ii) a CDRL2 selected from thegroup consisting of SEQ ID NO: 5, 17, 29, 34, 41, 65, 77, 101, 113, 130,131 and 137; (iii) a CDRL3 selected from the group consisting of SEQ IDNO: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114, 126, and 138; and (iv)a CDRL of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than four amino acids;or (C) one or more heavy chain CDRHs of (A) and one or more light chainCDRLs of (B).

In one embodiment, the isolated antigen-binding protein may comprise atleast one or two CDRH of the above-mentioned (A) and at least one or twoCDRL of the above-mentioned (B). In yet another aspect, the isolatedantigen-binding protein includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 andCDRL3.

In addition, the CDRH of the above-mentioned (A) is further selectedfrom the group consisting of: (i) a CDRH1 selected from the groupconsisting of SEQ ID NO: 10, 22, 70 94 and 142; (ii) a CDRH2 selectedfrom the group consisting of SEQ ID NO: 11, 23, 28, 35, 47, 59, 71, 95,106, 119 and 143; (iii) a CDRH3 selected from the group consisting ofSEQ ID NO: 12, 24, 36, 48, 60, 72, 83, 96, 108, 120, 132, and 144; and(iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than two amino acids;the CDRH of the above-mentioned (B) is selected from the groupconsisting of: (i) a CDRL1 selected from the group consisting of SEQ IDNO: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118, 124, 125 and 136;(ii) a CDRL2 selected from the group consisting of SEQ ID NO: 5, 17, 29,34, 41, 65, 77, 101, 113, 130, 131 and 137; (iii) a CDRL3 selected fromthe group consisting of SEQ ID NO: 6, 18, 42, 46, 66, 78, 84, 89, 90,102, 114, 126, and 138; and (iv) a CDRL of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids; or (C) one or more heavy chain CDRHs of(A) and one or more light chain CDRLs of (B).

In yet another embodiment, the isolated antigen-binding protein maycomprise (A) a CDRH selected from the group consisting of (i) a CDRH1selected from the group consisting of SEQ ID NO: 10, 22, 70 94 and 142;(ii) a CDRH2 selected from the group consisting of SEQ ID NO: 11, 23,28, 35, 47, 59, 71, 95, 106, 119 and 143; (iii) a CDRH3 selected fromthe group consisting of SEQ ID NO: 12, 24, 36, 48, 60, 72, 83, 96, 108,120, 132, and 144; (B) a CDRL selected from the group consisting of (i)a CDRL1 selected from the group consisting of SEQ ID NO: 4, 16, 30, 40,52, 64, 88, 100, 107, 112, 118, 124, 125 and 136; (ii) a CDRL2 selectedfrom the group consisting of SEQ ID NO: 5, 17, 29, 34, 41, 65, 77, 101,113, 130, 131 and 137; (iii) a CDRL3 selected from the group consistingof SEQ ID NO: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114, 126, and 138;or (C) one or more heavy chain CDRHs of (A) and one or more light chainCDRLs of (B). In one embodiment, the isolated antigen-binding proteinmay include (A) a CDRH1 selected from the group consisting of SEQ ID NO:10, 22, 70 94 and 142; a CDRH2 selected from the group consisting of SEQID NO: 11, 23, 28, 35, 47, 59, 71, 95, 106, 119 and 143; a CDRH3selected from the group consisting of SEQ ID NO: 12, 24, 36, 48, 60, 72,83, 96, 108, 120, 132, and 144; and (B) a CDRL1 selected from the groupconsisting of SEQ ID NO: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118,124, 125 and 136; a CDRL2 selected from the group consisting of SEQ IDNO: 5, 17, 29, 34, 41, 65, 77, 101, 113, 130, 131 and 137; and CDRL3selected from the group consisting of SEQ ID NO: 6, 18, 42, 46, 66, 78,84, 89, 90, 102, 114, 126, and 138. In another embodiment, the variableheavy chain (VH) has at least 90% sequence identity with an amino acidsequence selected from the group consisting of SEQ ID NO: 9, 21, 33, 45,57, 69, 81, 93, 105, 117, 129, and 141, and/or the variable light chain(VL) has at least 90% sequence identity with an amino acid sequenceselected from the group consisting of SEQ ID NO: 3, 15, 27, 39, 51, 63,75, 87, 99, 111, 123, and 135. In a further embodiment, the VH isselected from the group consisting of SEQ ID NO: 9, 21, 33, 45, 57, 69,81, 93, 105, 117, 129, and 141, and/or the VL is selected from the groupconsisting of SEQ ID NO: 3, 15, 27, 39, 51, 63, 75, 87, 99, 111, 123,and 135.

In another aspect, an isolated antigen binding protein is provided thatspecifically binds to an epitope containing GM-CSF sequences wherein theantibody binding to GM-CSF antagonizes activation of GM-CSF mediatedsignaling in a cell containing a GM-CSFR.

In yet another aspect, an isolated antigen binding protein is providedthat binds GM-CSF that comprises: (A) one or more heavy chain CDRs(CDRHs) selected from the group consisting of (i) a CDRH1 with at least80% identify to a CDRH1 selected from the group consisting of SEQ ID NO:10, 22, 70 94 and 142; (ii) a CDRH2 with at least 80% identity to aCDRH2 selected from the group consisting of SEQ ID NO: 11, 23, 28, 35,47, 59, 71, 95, 106, 119 and 143; (iii) a CDRH3 with at least 80%identity to a CDRH3 selected from the group consisting of SEQ ID NO: 12,24, 36, 48, 60, 72, 83, 96, 108, 120, 132, and 144; (B) one or morelight chain CDRs (CDRLs) selected from the group consisting of: (i) aCDRL1 that is 80% identical to a CDRL1 selected from the groupconsisting of SEQ ID NO: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118,124, 125 and 136; (ii) a CDRL2 that is 80% identical to a CDRL2 selectedfrom the group consisting of SEQ ID NO: 5, 17, 29, 34, 41, 65, 77, 101,113, 130, 131 and 137; (iii) a CDRL3 that is 80% identical to a CDRL3selected from the group consisting of SEQ ID NO: 6, 18, 42, 46, 66, 78,84, 89, 90, 102, 114, 126, and 138; or (C) one or more heavy chain CDRHsof (A) and one or more light chain CDRLs of (B). In one embodiment, theisolated antigen-binding protein includes (A) one or more CDRHs selectedfrom the group consisting of: (i) a CDRH1 with at least 90% identity toa CDRH1 selected from the group consisting of SEQ ID NO: 10, 22, 70 94and 142; (ii) a CDRH2 with at least 90% identity to a CDRH2 selectedfrom the group consisting of SEQ ID NO: 11, 23, 28, 35, 47, 59, 71, 95,106, 119 and 143; (iii) a CDRH3 with at least 90% identity to a CDRH3selected from the group consisting of SEQ ID NO: 12, 24, 36, 48, 60, 72,83, 96, 108, 120, 132, and 144; (B) one or more CDRLs selected from thegroup consisting of: (i) a CDRL1 that is at least 90% identical to aCDRL1 selected from the group consisting of SEQ ID NO: 4, 16, 30, 40,52, 64, 88, 100, 107, 112, 118, 124, 125 and 136; (ii) a CDRL2 that isat least 90% identical to a CDRL2 selected from the group consisting ofSEQ ID NO: 5, 17, 29, 34, 41, 65, 77, 101, 113, 130, 131 and 137; (iii)a CDRL3 that is at least 90% identical to a CDRL3 selected from thegroup consisting of SEQ ID NO: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102,114, 126, and 138; or (C) one or more heavy chain CDRHs of (A) and oneor more light chain CDRLs of (B).

In a further aspect, there is a provision of an isolated antigen-bindingprotein that binds GM-CSF, the antigen-binding protein including A) aheavy chain complementary determining region (CDRH) selected from thegroup consisting of (i) a CDRH3 selected from the group consisting ofSEQ ID NOs: 12, 24, 36, 48, 60, 72, 83, 96, 108, 120, 132, and 144; (ii)a CDRH3 that differs in amino acid sequence from the CDRH3 of (i) by anamino acid addition, deletion or substitution of not more than two aminoacids; (iii) a CDRH3 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆X₇X₈FDX₉ (SEQ ID NO: 83) wherein X₁ isselected from the group consisting of E and no amino acid, X₂ isselected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of P, D and G, X₄ is selected fromthe group consisting of Y, W, R and K, X₅ is selected from the groupconsisting of S, W, F, and T, X₆ is selected from the group consistingof Y and L, X₇ is selected from the group consisting of D and G, X₈ isselected from the group consisting of Y, no amino acid, and A, and X₉ isselected from the group consisting of M, T, and V; and/or B) a lightchain complementary determining region (CDRL) selected from the groupconsisting of (i) a CDRL3 selected from the group consisting of SEQ IDNOs: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114, 126, and 138 (ii) aCDRL3 that differs in amino acid sequence from the CDRL3 of (i) by anamino acid addition, deletion or substitution of not more than two aminoacids; and iii) a CDRL3 amino acid sequence selected from the groupconsisting of X₁QX₂X₃X₄X₅X₆X₇T (SEQ ID NO: 84) wherein X1 is selectedfrom the group consisting of Q and L, X₂ is selected from the groupconsisting of Y and S, X₃ is selected from the group consisting of D, Gand F, X₄ is selected from the group consisting of R, T, and S, X₅ isselected from the group consisting of S and V, X₆ is selected from thegroup consisting of F and P, and X₇ is selected from the groupconsisting of R and W; and X₁X₂X₃X₄DSSNX₅X₆X₇ (SEQ ID NO: 89) wherein X₁is selected from the group consisting of S and A, X₂ is selected fromthe group consisting of S and A, X₃ is selected from the groupconsisting of W and F, X₄ is selected from the group consisting of D andT, X₅ is selected from the group consisting of G, W, and no amino acid,X6 is selected from the group consisting of V, L, and P, and X₆ isselected from the group consisting of V and no amino acid.

Within another embodiment, the antigen binding protein furthercomprising: A) a CDRH selected from the group consisting of: (i) a CDRH1selected from the group consisting of SEQ ID NOs: 10, 22, 70 94 and 142;(ii) a CDRH1 that differs in amino acid sequence from the CDRH1 of (i)by an amino acid addition, deletion or substitution of not more than twoamino acids; (iii) a CDRH1 amino acid sequence selected from the groupconsisting of X₁X₂GX₃X₄XFX₅X₆YX₇X₈X₉ (SEQ ID NO: 94) wherein X₁ isselected from the group consisting of G and no amino acid, X₂ isselected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of Y and F, X₄ is selected from thegroup consisting of T and S, X₅ is selected from the group consisting ofT, S and G, X₆ is selected from the group consisting of G and S, X₇ isselected from the group consisting of Y and G, X₈ is selected from thegroup consisting of I and M, and X₉ is selected from the groupconsisting of H and S, or (iv) a CDRH2 selected from the groupconsisting of SEQ ID NOs: 5, 17, 29, 34, 41, 65, 77, 101, 113, 130, 131and 137; (v) a CDRH2 that differs in amino acid sequence from the CDRH2of (iv) by an amino acid addition, deletion or substitution of not morethan two amino acids; or (vi) a CDRH2 amino acid sequence consisting ofX₁X₂X₃X₄X₅X₆GX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅G (SEQ ID NO: 106) wherein X₁ isselected from the group consisting of W and no amino acid, X₂ isselected from the group consisting of I and Y, X₃ is selected from thegroup consisting of N, S and I, X₄ is selected from the group consistingof P, A and Y, X₅ is selected from the group consisting of N and Y, X₆is selected from the group consisting of S and N, X₇ is selected fromthe group consisting of G and N, X₈ is selected from the groupconsisting of T and R, X₉ is selected from the group consisting of N andD, X₁₀ is selected from the group consisting of Y and S, X₁₁ is selectedfrom the group consisting of A and N, X₁₂ is selected from the groupconsisting of Q and R, X₁₃ is selected from the group consisting of Kand R, X₁₄ is selected from the group consisting of F and L, and X₁₅ isselected from the group consisting of Q, K and R; or B) a CDRL selectedfrom the group consisting of: (i) a CDRL1 selected from the groupconsisting of SEQ ID NOs: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118,124, 125 and 136; (ii) a CDRL1 that differs in amino acid sequence fromthe CDRL1 of (i) by an amino acid addition, deletion or substitution ofnot more than two amino acids; (iii) a CDRL1 amino acid sequenceselected from the group consisting of KSSQSX₁XLYSSX₂NX₃NX₄LX₅ (SEQ IDNO: 107) wherein X₁ is selected from the group consisting of V and I, X₂is selected from the group consisting of S and N, X₃ is selected fromthe group consisting of E and K, X₄ is selected from the groupconsisting of Y and F, and X₅ is selected from the group consisting of Tand A; RASX₁X₂X₃X₄X₅X₆YX₇X₈ (SEQ ID NO: 118) wherein X₁ is selected fromthe group consisting of Q and P, X₂ is selected from the groupconsisting of S and Y, X₃ is selected from the group consisting of V, Land I, X₄ is selected from the group consisting of S and C, X₅ isselected from the group consisting of S and N, X₆ is selected from thegroup consisting of S, I, T and no amino acid, X₇ is selected from thegroup consisting of F and L, and X₈ is selected from the groupconsisting of A and N; or X₁X₂X₃X₄X₅X₆YX₇X₈X₉X₁₀NX₁₁VX₁₂ (SEQ ID NO:125) wherein X₁ is selected from the group consisting of I, S and T, X₂is selected from the group consisting of R and G, X₃ is selected fromthe group consisting of T and S, X₄ is selected from the groupconsisting of R and S, X₅ is selected from the group consisting of G andS, X₆ is selected from the group consisting of S, H and D, X₇ isselected from the group consisting of I and V, X₈ is selected from thegroup consisting of A and G, X₉ is selected from the group consisting ofno amino acid and G, X₁₀ is selected from the group consisting of S andY, X₁₁ is selected from the group consisting of Y and T, and X₁₂ isselected from the group consisting of Q, N and S; or (iv) a CDRL2selected from the group consisting of SEQ ID NOs: 5, 17, 29, 34, 41, 65,77, 101, 113, 130, 131 and 137; (v) a CDRL2 that differs in amino acidsequence from the CDRL2 of (iv) by an amino acid addition, deletion orsubstitution of not more than two amino acids; or (vi) a CDRL2 aminoacid sequence selected from the group consisting of X₁X₂X₃X₄X₅X₆X₇ (SEQID NO: 130) wherein X₁ is selected from the group consisting of G, T andW, X2 is selected from the group consisting of T and A, X3 is selectedfrom the group consisting of S and A, X4 is selected from the groupconsisting of S and T, X5 is selected from the group consisting of R andL, X6 is selected from the group consisting of A, E and Q, and X7 isselected from the group consisting of T and S; or X₁X₂X₃X₄RPS (SEQ IDNO: 131) wherein X1 is selected from the group consisting of E and S, X2is selected from the group consisting of D, V and N, X3 is selected fromthe group consisting of D, S and N, and X4 is selected from the groupconsisting of Q, G and H.

In one aspect, the isolated antigen-binding proteins provided herein canbe a monoclonal antibody, a polyclonal antibody, a recombinant antibody,a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof. In anotherembodiment, the antibody fragment of the isolated antigen-bindingproteins can be an Fab fragment, an Fab′ fragment, an F(ab′)₂ fragment,an Fv fragment, a diabody, or a single chain antibody molecule. In afurther embodiment, the isolated antigen binding protein is a humanantibody and can be an IgG1, IgG2, IgG3, or IgG4.

In yet another aspect, the isolated antigen-binding protein can becoupled to a labeling group and can compete for binding to theextracellular portion of human GM-CSF with an antigen binding protein ofone of the isolated antigen-binding proteins provided.

In yet another aspect, the isolated antigen binding protein thatcompetes for binding to the receptor interacting portion of human GM-CSFwith an antigen binding protein as provided herein. In one embodiment,the antigen binding protein is a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, a chimeric antibody, a multispecific antibody, or an antibodyfragment thereof. In related embodiments are provided human andmonoclonal antibodies and antigen fragments including a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment, a Fv fragment, a diabody, or a singlechain antibody molecule. In other embodiments the antigen bindingprotein is of the IgG1-, IgG2- IgG3- or IgG4-type. Further provided isthe isolated antigen binding protein coupled to a labeling group.

In yet another aspect, the invention further contemplates inhibitingGM-CSF activity to limit signals that cause or effect differentiation,survival, proliferation and activation of myeloid lineage cellsincluding macrophages/monocytes, dendritic cells (DCs), neutrophils andeosinophils, and/or differentiation and/or activation of DCs. Inaddition, the invention contemplates inhibiting GM-CSF activity to limitmonocyte-derived macrophages (Type 1 macrophages) from producing highlevels of proinflammatory cytokines such as IL-23.

In a further aspect, also provided are isolated polynucleotides thatencode the antigen-binding proteins that bind to GM-CSF, wherein theisolated polynucleotides are operably-linked to a control sequence.

In another aspect, also provided are expression vectors and host cellstransformed or transfected with the expression vectors that comprisingthe aforementioned isolated polynucleotides that encode antigen-bindingproteins that can bind to GM-CSF.

In another aspect, also provided are methods of preparing theantigen-binding proteins that includes the step of preparing the antigenbinding protein from a host cell that secretes the antigen-bindingprotein.

In yet another aspect, a pharmaceutical composition is providedcomprising at least one of the aforementioned antigen-binding proteinsprovided and a pharmaceutically acceptable excipient. In one embodiment,the pharmaceutical composition may comprise an additional active agentthat is selected from the group consisting of a radioisotope,radionuclide, a toxin, or a therapeutic and a chemotherapeutic group.

In yet another aspect, a method is provided for treating or preventing acondition associated with GM-CSF in a patient, comprising administeringto a patient an effective amount of at least one isolatedantigen-binding protein. In one embodiment the condition is selectedfrom the group consisting of rheumatic disorders, autoimmune disorders,hematological disorders, oncological disorders, inflammatory disorders,degenerative conditions of the nervous system, gastrointestinal,gastrourinary disorders, endocrine disorders and the like. In oneembodiment, the condition is a disorder or disease that is selected fromthe group consisting of multiple sclerosis (MS), rheumatoid arthritis(RA), asthma, psoriasis, atopic dermatitis and sarcoidosis. In anotherembodiment is included treatment with an isolated antigen-bindingprotein alone or as a combination therapy. In yet another embodiment thecondition is selected from breast cancer, prostate cancer, colorectalcancer, endometrial adenocarcinoma, leukemia, lymphoma, melanoma,gastric cancer, astrocytic cancer, endometrial cancer, cervical cancer,bladder cancer, renal cancer, and ovarian cancer.

In another aspect, the invention provides a method of inhibiting bindingof GM-CSF to the extracellular portion of GM-CSFR in a patientcomprising administering an effective amount of at least oneantigen-binding protein provided herein.

In yet another aspect, the invention provides a method of inhibitingphosphorylation of human GM-CSFR in a patient comprising administeringan effective amount of at least one antigen binding protein as describedherein.

In yet another aspect, the isolated antigen binding protein reducesmonocyte chemotaxis when administered to a patient. In one embodiment,the antigen binding protein inhibits monocyte migration. In yet anotherembodiment monocyte migration into tumors is inhibited when the isolatedantigen binding protein is administered to a patient.

Further provided, as yet another aspect is a method of treating multiplesclerosis comprising administering an isolated antigen-binding proteinas described herein.

Also contemplated are conditions were multiple sclerosis isrelapsing-remitting multiple sclerosis, progressive-relapsing multiplesclerosis, primary-progressive multiple sclerosis or secondaryprogressive multiple sclerosis.

In one aspect also provided is a method of treating rheumatoid arthritiscomprising administering an isolated antigen-binding protein asdescribed herein.

In additional aspects are provided isolated antigen binding proteinsthat are cross-reactive with cynomologous GM-CSF within 1 log of humanGM-CSF and that bind GM-CSF with an IC₅₀ of <1 nM as measured in aGM-CSF dependent assay.

These and other aspects will be described in greater detail herein. Eachof the aspects provided can encompass various embodiments providedherein. It is therefore anticipated that each of the embodimentsinvolving one element or combinations of elements can be included ineach aspect described. Other features, objects, and advantages of thedisclosed are apparent in the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-d: A) Prophylactic administration of anti-murine GM-CSF MAb inactive EAE delayed onset and reduced incidence of disease. To induceactive SJL/PLP₁₃₉₋₁₅₁ EAE, 11 mice were given 250 μg PLP₁₃₉₋₁₅₁+CFAsubcutaneous and subjected to a three week assessment. Eleven mice pergroup were given 500 μg anti-murine GM-CSF mAb, isotype control mAb orPBS on day of immunization. Daily weights and scoring were taken.Clinical scoring 0: no disease; 1: limp tail; 2: slight impairment ofrighting reflex or abnormal gait; 3: severe hind limb weakness, partialhind limb paralysis; 4: complete hind limb paralysis, mobile usingforelimbs. Anti-GM-CSF mAb shows delay of onset compared to controls,with incidence at 45% compared to 91-100% in controls.

B and C) Prophylactic administration of anti-GM-CSF mAb prevented weightloss (B) and reduced mean clinical score (C).

D) Results from single dose of 500 μg anti-mGM-CSF mAb, isotype controlmAb or PBS on day of disease onset, n=14 mice. Therapeuticadministration of anti-GM-CSF mAb in active EAE reduced mean clinicalscore. P<0.05 vs isotype control or PBS.

FIGS. 2 a-c: A) Therapeutic administration of anti-mGM-CSF mAb in activeEAE accelerated recovery. Recovery=decrease of ≧1 full score≧2dconsecutively to score of ≦1.

B) Therapeutic anti-mGM-CSF mAb treatment on day of disease onset (day13 post-immunization) reduced CNS inflammation compared to mice treatedwith anti-mGM-CSF mAb, isotype control mAb or PBS control.

C and D) Prophylactic or therapeutic administration of anti-mGM-CSF mAbin adoptive transfer EAE ameliorated disease. In the adoptive transferEAE model, 15 mice were given 100 μg PLP₁₃₉₋₁₅₁+CFA and, lymph node wereharvested on day 10 post-immunization stimulated with PLP peptide 4 daysin vitro and injected into recipient mice. Mice were subjected to threeweek assessment of weight and clinical score. FIG. C shows treatment onday of cell transfer, FIG. D shows treatment on day of EAE onset.

FIG. 3: Representative TF-1 Stat5 phosphorylation assay showinganti-GM-CSF mAb inhibition or 0.4 ng/ml rhGM-CSF-Ile.

FIG. 4: Histogram showing distribution of inhibition of rhGM-CSF-Ile inthe TF-1 STAT5 phosphorylation assay my hybridoma supernatant from E.coli rhGM-CSF immunized mice.

FIG. 5: Representative AML-5 proliferation assay showing anti-GM-CSF mAbinhibition of 0.15 ng/ml rhGM-CSF-Ile.

FIGS. 6 a-f: Inhibition of GM-CSF and CSF-1 by mAb purified fromhybridoma supernatants in the AML-5 proliferation assay.

FIGS. 7 a-b: Inhibition of GM-CSF and IL-3 by mAb purified fromhybridoma supernatants in the TF-1 Stat5 phosphorylation assay.

FIGS. 8 a-b: Results from ELISA measuring binding of hybridomasupernatant anti-GM-CSF mAb or recombinant anti-GM-CSF mAb to GM-CSFfrom various species.

FIG. 9: Inhibition of human and cyno GM-CSF by recombinant mAb (2^(nd)TT and 2^(nd) SCL) in the AML-5 proliferation assay.

FIG. 10: Inhibition of human and cyno GM-CSF by recombinant mAb (2^(nd)TT and 2^(nd) SCL) in the TF-1 Stat-5 phosphorylation assay.

FIG. 11: Inhibition of native human and cyno lung-derived GM-CSF byrecombinant mAb (1^(st) TT) in the TF-1 Stat5 phosphorylation assay.

FIG. 12: Inhibition of native cyno PBMC-derived GM-CSF by recombinantmAb (2^(nd) SCL) IgG B in the TF-1 Stat5 phosphorylation assay.

FIG. 13: Inhibition of human GM-CSF by recombinant mAb (2^(nd) TT) inthe human monocyte assay.

FIG. 14: Inhibition of GM-CSF-induced production of MIP-1b and ENA78 inwhole blood by recombinant mAb (2^(nd) SCL) IgG B.

FIGS. 15 a-b: mAb purified from hybridoma supernatants does not inhibityeast-derived rhGM-CSF (Leukine®) in the AML-5 proliferation assay (a)or the monocyte assay (b).

FIG. 16: mAb neutralized E. coli-derived rhGM-CSF but not yeast-derivedrhGM-CSF (Leukine®) or unglycosylated E. coli-derived rhGM-CSF-R23L.

DETAILED DESCRIPTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the disclosed, which is defined solely by the claims.

The methods and techniques of the present application are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990).

The term “polynucleotide” includes both single-stranded anddouble-stranded nucleic acids and includes genomic DNA, RNA, mRNA, cDNA,or synthetic origin or some combination thereof which is not associatedwith sequences normally found in nature. Isolated polynucleotidescomprising specified sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or may include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or may include vector sequences. Thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 100 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidesmay be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene. Oligonucleotides may be sense orantisense oligonucleotides. An oligonucleotide can include a detectablelabel, such as a radiolabel, a fluorescent label, a hapten or anantigenic label, for detection assays. Oligonucleotides may be used, forexample, as PCR primers, cloning primers or hybridization probes.

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, control sequences for prokaryotesmay include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes mayinclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” can includeleader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell. The term “expression vector” or“expression construct” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control (in conjunction with the host cell) expression ofone or more heterologous coding regions operatively linked thereto. Anexpression construct may include, but is not limited to, sequences thataffect or control transcription, translation, and, if introns arepresent, affect RNA splicing of a coding region operably linked thereto.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions. For example, a control sequence, e.g., apromoter, in a vector that is “operably linked” to a protein codingsequence are arranged such that normal activity of the control sequenceleads to transcription of the protein coding sequence resulting inrecombinant expression of the encoded protein.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The terms “polypeptide” or “protein” means a macromolecule having theamino acid sequence of a native protein, that is, a protein produced bya naturally-occurring and non-recombinant cell; or it is produced by agenetically-engineered or recombinant cell, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The term also includes amino acidpolymers in which one or more amino acids are chemical analogs of acorresponding naturally-occurring amino acid and polymers. The terms“polypeptide” and “protein” encompass GM-CSF antigen-binding proteins,antibodies, or sequences that have deletions from, additions to, and/orsubstitutions of one or more amino acid of antigen-binding protein. Theterm “polypeptide fragment” refers to a polypeptide that has anamino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion as compared with the full-length native protein. Suchfragments may also contain modified amino acids as compared with thenative protein. In certain embodiments, fragments are about five to 500amino acids long. For example, fragments may be at least 5, 6, 8, 10,14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 aminoacids long. Useful polypeptide fragments include immunologicallyfunctional fragments of antibodies, including binding domains. In thecase of a GM-CSF-binding antibody, useful fragments include but are notlimited to a CDR region, a variable domain of a heavy or light chain, aportion of an antibody chain or just its variable region including twoCDRs, and the like.

The term “isolated protein” refers to a protein that is purified fromproteins or polypeptides or other contaminants that would interfere withits therapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antigen binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins. A “derivative” of a polypeptide is apolypeptide (e.g., an antigen binding protein, or an antibody) that hasbeen chemically modified in some manner distinct from insertion,deletion, or substitution variants, e.g., via conjugation to anotherchemical moiety.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature.

An “antigen binding protein” as used herein means a protein thatspecifically binds a specified target antigen; the antigen of theprovided is GM-CSF, or human GM-CSF.

An antigen binding protein is said to “specifically bind” its targetantigen when the dissociation constant (K_(d)) is ≦10⁻⁸ M. The antibodyspecifically binds antigen with “high affinity” when the K_(d) is≦5×10⁻⁹ M, and with “very high affinity” when the K_(d) is ≦5×10⁻¹⁰ M.In one embodiment, the antibody has a K_(d) of ≦10⁻⁹ M and an off-rateof about 1×10⁻⁴/sec. In one embodiment, the off-rate is <1×10⁻⁸. Inother embodiments, the antibodies will bind to GM-CSF, or human GM-CSFwith a K_(d) of between about 10⁻⁸ M and 10⁻¹⁰ M, and in yet anotherembodiment it will bind with a K_(d)≦2×10⁻¹⁰.

“Antigen binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen. For example, that portionof an antigen binding protein that contains the amino acid residues thatinteract with an antigen and confer on the antigen binding protein itsspecificity and affinity for the antigen is referred to as “antigenbinding region.” An antigen binding region typically includes one ormore “complementary binding regions” (“CDRs”). Certain antigen bindingregions also include one or more “framework” regions. A “CDR” is anamino acid sequence that contributes to antigen binding specificity andaffinity. “Framework” regions are can aid in maintaining the properconformation of the CDRs to promote binding between the antigen bindingregion and an antigen.

In certain aspects, recombinant antigen binding proteins that bindGM-CSF protein, or human GM-CSF, are provided. In this context, a“recombinant protein” is a protein made using recombinant techniques,i.e., through the expression of a recombinant nucleic acid as describedherein. Methods and techniques for the production of recombinantproteins are well known in the art.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An“antibody” as such is a species of an antigen binding protein. An intactantibody generally will comprise at least two full-length heavy chainsand two full-length light chains, but in some instances may includefewer chains such as antibodies naturally occurring in camelids whichmay comprise only heavy chains. Antibodies may be derived solely from asingle source, or may be “chimeric,” that is, different portions of theantibody may be derived from two different antibodies as describedfurther below. The antigen binding proteins, antibodies, or bindingfragments may be produced in hybridomas, by recombinant DNA techniques,or by enzymatic or chemical cleavage of intact antibodies. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient sequence to confer binding specificity. In atypical mammalian antibody, one will find a full-length light chain thatincludes a variable region domain, V_(L), and a constant region domain,C_(L), where the variable region domain of the light chain is toward theamino-terminus of the polypeptide. Typical human antibody light chainsinclude kappa chains or lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A mammalian full-length heavy chain antibody typicallyincludes a variable region domain, V_(H), and three constant regiondomains, C_(H)1, C_(H)2, and C_(H)3. The V_(H) domain is toward theamino-terminus of the polypeptide, and the C_(H) domains are toward thecarboxyl-terminus, with the C_(H)3 being closest to the carboxy-terminusof the polypeptide. Human heavy chains typically may be of isotypes thatinclude IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA(including IgA1 and IgA2 subtypes), IgM and IgE.

The term “functional fragment” (or simply “fragment”) of an antibody orimmunoglobulin chain (heavy or light chain), as used herein, is anantigen binding protein comprising a portion (regardless of how thatportion is obtained or synthesized) of an antibody that lacks at leastsome of the amino acids present in a full-length chain but which iscapable of specifically binding to an antigen. Such fragments arebiologically active in that they bind specifically to the target antigenand can compete with other antigen binding proteins, including intactantibodies, for specific binding to a given epitope. In one aspect, sucha fragment will retain at least one CDR present in the full-length lightor heavy chain, and in some embodiments will comprise a single heavychain and/or light chain or portion thereof. These biologically activefragments may be produced by recombinant DNA techniques, or may beproduced by enzymatic or chemical cleavage of antigen binding proteins,including intact antibodies. Immunologically functional immunoglobulinfragments include, but are not limited to, Fab, Fab′, F(ab′)₂, Fv,domain antibodies and single-chain antibodies, and may be derived fromany mammalian source, including but not limited to human, mouse, rat,camelid or rabbit. It is contemplated further that a functional portionof the antigen binding proteins disclosed herein, for example, one ormore CDRs, could be covalently bound to a second protein or to a smallmolecule to create a therapeutic agent directed to a particular targetin the body, possessing bifunctional therapeutic properties, or having aprolonged serum half-life.

An “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of an Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the VH domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. An F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in PCT Publication No.WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antigen binding protein” or “bivalent antibody” comprisestwo antigen binding sites. In some instances, the two binding sites havethe same antigen specificities. Bivalent antigen binding proteins andbivalent antibodies may be bispecific, see, infra.

A multispecific antigen binding protein” or “multispecific antibody” isone that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein or antibody,respectively, having two different antigen binding sites. Bispecificantigen binding proteins and antibodies are a species of multispecificantigen binding protein antibody and may be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp.Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.The two binding sites of a bispecific antigen binding protein orantibody will bind to two different epitopes, which may reside on thesame or different protein targets.

The term “neutralizing antigen binding protein” or “neutralizingantibody” refers to an antigen binding protein or antibody,respectively, that binds to a ligand, prevents binding of the ligand toits binding partner and interrupts the biological response thatotherwise would result from the ligand binding to its binding partner.In assessing the binding and specificity of an antigen binding protein,e.g., an antibody or immunologically functional fragment thereof, anantibody or fragment will substantially inhibit binding of a ligand toits binding partner when an excess of antibody reduces the quantity ofbinding partner bound to the ligand by at least about 20%, 30%, 40%,50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in anin vitro competitive binding assay). In the case of a GM-CSF antigenbinding proteins, such a neutralizing molecule will diminish the abilityof GM-CSF to bind GM-CSFR.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins or neutralizing antibodies)that compete for the same epitope means competition between antigenbinding proteins is determined by an assay in which the antigen bindingprotein (e.g., antibody or immunologically functional fragment thereof)under test prevents or inhibits specific binding of a reference antigenbinding protein (e.g., a ligand, or a reference antibody) to a commonantigen (e.g., a GM-CSF or a fragment thereof). Numerous types ofcompetitive binding assays can be used, for example: solid phase director indirect radioimmunoassay (RIA), solid phase direct or indirectenzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahliet al., 1983, Methods in Enzymology 9:242-253); solid phase directbiotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase directlabeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, ALaboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer etal., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assayinvolves the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabelled test antigen binding protein anda labeled reference antigen binding protein.

Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the test antigenbinding protein. Usually the test antigen binding protein is present inexcess. Antigen binding proteins identified by competition assay(competing antigen binding proteins) include antigen binding proteinsbinding to the same epitope as the reference antigen binding proteinsand antigen binding proteins binding to an adjacent epitope sufficientlyproximal to the epitope bound by the reference antigen binding proteinfor steric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the examples herein.Usually, when a competing antigen binding protein is present in excess,it will inhibit specific binding of a reference antigen binding proteinto a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or75%. In some instance, binding is inhibited by at least 80%, 85%, 90%,95%, or 97% or more.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof), and additionally capable of being used inan animal to produce antibodies capable of binding to that antigen. Anantigen may possess one or more epitopes that are capable of interactingwith different antigen binding proteins, e.g., antibodies.

The term “epitope” includes any determinant capable of specificallybinding to an antigen binding protein. An epitope is a region of anantigen that is bound by an antigen binding protein that specificallytargets that antigen, and when the antigen is a protein, includesspecific amino acids that contact the antigen binding protein. Mostoften, epitopes reside on proteins which are understood to include nonamino acid post-translational modifications, but in some instances mayreside on other kinds of molecules, such as nucleic acids. Epitopes mayinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and may havespecific three dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially recognize an epitope on the targetantigen in a complex mixture of proteins and/or macromolecules.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more polynucleotides, asdetermined by aligning and comparing the sequences. “Percent identity”means the percent of identical residues between the amino acids ornucleotides in the compared molecules and is calculated based on thesize of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as 3× theaverage diagonal, wherein the “average diagonal” is the average of thediagonal of the comparison matrix being used; the “diagonal” is thescore or number assigned to each perfect amino acid match by theparticular comparison matrix) and a gap extension penalty (which isusually 1/10 times the gap opening penalty), as well as a comparisonmatrix such as PAM 250 or BLOSUM 62 are used in conjunction with thealgorithm. In certain embodiments, a standard comparison matrix (see,Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) isalso used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition.

In certain embodiments, an essentially homogeneous substance has beenpurified to such a degree that contaminating species cannot be detectedin the composition by conventional detection methods and thus thecomposition consists of a single detectable macromolecular species.

The term “therapeutically effective amount” refers to the amount of aGM-CSF antigen binding protein determined to produce any therapeuticresponse in a mammal. Such therapeutically effective amounts are readilyascertained by one of ordinary skill in the art.

“Amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S.Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, Mass.(1991). Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as [alpha]-,[alpha]-disubstituted amino acids, N-alkyl amino acids, and otherunconventional amino acids may also be suitable components forpolypeptides. Examples of unconventional amino acids include:4-hydroxyproline, [gamma]-carboxyglutamate,[epsilon]-N,N,N-trimethyllysine, [epsilon]-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, [sigma]-N-methylarginine, and other similar amino acidsand imino acids (e.g., 4-hydroxyproline). In the polypeptide notationused herein, the left-hand direction is the amino terminal direction andthe right-hand direction is the carboxyl-terminal direction, inaccordance with standard usage and convention.

A. General Overview

Antigen-binding proteins that bind GM-CSF protein, in particular humanGM-CSF (hGM-CSF) protein are provided herein. The antigen bindingproteins provided are polypeptides into which one or more complementarydetermining regions (CDRs), as described herein, are embedded and/orjoined. In some antigen binding proteins, the CDRs are embedded into a“framework” region, which orients the CDR(s) such that the properantigen binding properties of the CDR(s) is achieved. In general,antigen binding proteins that are provided can interfere with, block,reduce or modulate the interaction between GM-CSFR and GM-CSF.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. The various structures are further describedherein.

The antigen binding proteins provided herein have been demonstrated tobind to certain epitopes of GM-CSF, in particular human GM-CSF. As aconsequence, the antigen binding proteins provided herein are capable ofinhibiting GM-CSF activity. In particular, antigen binding proteinsbinding to these epitopes inhibit, inter alia, induction of GM-CSFRsignaling, GM-CSF induced cell growth or differentiation, and otherphysiological effects induced by GM-CSF upon binding to GM-CSFR.

The antigen binding proteins that are disclosed herein have a variety ofutilities. Some of the antigen binding proteins, for instance, areuseful in specific binding assays, affinity purification of GM-CSF, inparticular hGM-CSF or its ligands and in screening assays to identifyother antagonists of GM-CSF activity. Some of the antigen-bindingproteins are useful for inhibiting binding of GM-CSFR to GM-CSF, orinhibiting autophosphorylation of GM-CSF.

The antigen-binding proteins can be used in a variety of treatmentapplications, as explained herein. For example, certain GM-CSFantigen-binding proteins are useful for treating conditions associatedwith GM-CSF, such as reducing monocyte chemotaxis in a patient,inhibiting monocyte migration into tumors, or inhibiting accumulation oftumor associated macrophage in a tumor, as is further described herein.Other uses for the antigen binding proteins include, for example,diagnosis of GM-CSF-associated diseases or conditions and screeningassays to determine the presence or absence of GM-CSF. Some of theantigen binding proteins described herein are useful in treatingconsequences, symptoms, and/or the pathology associated with GM-CSFactivity. These include, but are not limited to, various types ofinflammatory disease.

B. GM-CSF Antigen Binding Proteins

A variety of selective binding agents useful for regulating the activityof GM-CSF are provided. These agents include, for instance, antigenbinding proteins that contain an antigen binding domain (e.g., singlechain antibodies, domain antibodies, immunoadhesions, and polypeptideswith an antigen binding region) and specifically bind to a GM-CSFpolypeptide, in particular human GM-CSF. Some of the agents, forexample, are useful in inhibiting the binding of GM-CSFR to GM-CSF, andcan thus be used to inhibit one or more activities associated withGM-CSF signaling.

In general, the antigen binding proteins that are provided typicallycomprise one or more CDRs as described herein (e.g., 1, 2, 3, 4, 5 or 6CDRs). In some instances, the antigen binding protein comprises (a) apolypeptide structure and (b) one or more CDRs that are inserted intoand/or joined to the polypeptide structure. The polypeptide structurecan take a variety of different forms. For example, it can be, orcomprise, the framework of a naturally occurring antibody, or fragmentor variant thereof, or may be completely synthetic in nature. Examplesof various polypeptide structures are further described below.

In certain embodiments, the polypeptide structure of the antigen bindingproteins is an antibody or is derived from an antibody, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the antigen binding protein is an immunological fragment ofan antibody (e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv). The variousstructures are further described and defined herein.

Certain of the antigen binding proteins as provided herein specificallybind to human GM-CSF. “Specifically binds” as used herein means theequilibrium dissociation constant is <10⁻⁸ to <10⁻¹⁰ M, alternatively<10⁻⁸ to <10⁻¹⁰ M.

In embodiments where the antigen binding protein is used for therapeuticapplications, an antigen binding protein can inhibit, interfere with ormodulate one or more biological activities of a GM-CSF. In this case, anantigen binding protein binds specifically and/or substantially inhibitsbinding of human GM-CSF to GM-CSFR when an excess of antibody reducesthe quantity of human GM-CSF bound to GM-CSFR, or vice versa, by atleast about 40%, 60%, 80%, 85%, or more (for example by measuringbinding in an in vitro competitive binding assay). GM-CSF has manydistinct biological effects, which can be measured in many differentassays in different cell types; examples of such assays are providedherein.

Some of the antigen binding proteins that are provided have thestructure typically associated with naturally occurring antibodies. Thestructural units of these antibodies typically comprise one or moretetramers, each composed of two identical couplets of polypeptidechains, though some species of mammals also produce antibodies havingonly a single heavy chain. In a typical antibody, each pair or coupletincludes one full-length “light” chain (in certain embodiments, about 25kDa) and one full-length “heavy” chain (in certain embodiments, about50-70 kDa). Each individual immunoglobulin chain is composed of several“immunoglobulin domains”, each consisting of roughly 90 to 110 aminoacids and expressing a characteristic folding pattern. These domains arethe basic units of which antibody polypeptides are composed. Theamino-terminal portion of each chain typically includes a variabledomain that is responsible for antigen recognition. The carboxy-terminalportion is more conserved evolutionarily than the other end of the chainand is referred to as the “constant region” or “C region”. Human lightchains generally are classified as kappa and lambda light chains, andeach of these contains one variable domain and one constant domain.Heavy chains are typically classified as mu, delta, gamma, alpha, orepsilon chains, and these define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subtypes, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes includeIgM, and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgAand IgD isotypes contain four heavy chains and four light chains; theIgG and IgE isotypes contain two heavy chains and two light chains; andthe IgM isotype contains five heavy chains and five light chains. Theheavy chain C region typically comprises one or more domains that may beresponsible for effector function. The number of heavy chain constantregion domains will depend on the isotype. IgG heavy chains, forexample, each contains three C region domains known as C_(H)1, C_(H)2and C_(H)3. The antibodies that are provided can have any of theseisotypes and subtypes. In certain embodiments, the GM-CSF antibody is ofthe IgG1, IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about twelve or more amino acids, with theheavy chain also including a “D” region of about ten more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press. The variable regions of each light/heavy chainpair typically form the antigen binding site.

1. Variable Domains of Antibodies

The various heavy chain and light chain variable regions provided hereinare depicted in TABLE 1. Each of these variable regions may be attachedto the above heavy and light chain constant regions to form a completeantibody heavy and light chain, respectively. Further, each of the sogenerated heavy and light chain sequences may be combined to form acomplete antibody structure.

Provided are antigen binding proteins that contain an antibody heavychain variable region selected from the group consisting of V_(H)1,V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, and V_(H)12, and/or an antibody light chain variable regionselected from the group consisting of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, V_(L)11, and V_(L)12,as shown in TABLE 1 below.

Antigen binding proteins of this type can generally be designated by theformula “V_(H)x/V_(L)y,” where “x” corresponds to the number of heavychain variable regions and “y” corresponds to the number of the lightchain variable regions as listed in TABLE 1:

TABLE 1 Exemplary VH and VL Chains Designation Amino Acid SequenceV_(H)1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHVVVRQAPGQGLEWMGWINPNSGGTNSAQKFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGGYSYGYFDYWGQG TLVTVSS [SEQ. ID. NO: 9] V_(H)2QVQLVQSGAEVKKPGASVKVSCKSSGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDKWLDGFDYWGQGTLV TVSSS [SEQ. ID. NO: 21] V_(H)3QVQLVQSGAAVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTASMELSRLRSDDTAVYFCARDRWLDAFDIWGQGTMV TVSS [SEQ. ID. NO: 33] V_(H)4QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQRFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARAPYDWTFDYWGQGTLV TVSS [SEQ. ID. NO: 45] V_(H)5QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGRNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDRWLDAFEIWGQGTMV TVSS [SEQ. ID. NO: 57] V_(H)6QVQLVQSGAEVKQPGASVKVSCEASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTDYAQKLQGRVTMTTDTSTSAAYMELRSLRSDDTAVYYCARQRYYYSMDVWGQGTTV TVSS [SEQ. ID. NO: 69] V_(H)7QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISSTAYMELWLRSDDTAVYYCARDRWLDAFDIWGQGTMV TVS [SEQ. ID. NO: 81] V_(H)8QVQLVQSGAEVKKPGASVKVSCKASGFTFSGYYMYWVRQAPGQGLEWMGWINPNSGGTNYARKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARRPWELPFDYWGQGTLV TVSS [SEQ. ID. NO:93] V_(H)9QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFKGRVTMTRDTSISTAHMELSRLRSDDTAVYYCVRNGDYVFTYFDYWGQGT LVTVSS [SEQ. ID. NO: 105]+V_(H)10 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFRGRVTMTRDTSISSTAYMELRLRSDDTAVYYCARFGYFGYYFDYWGQGTL VTVSS [SEQ. ID. NO: 117] V_(H)11QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFRGRVTMTRDTSISTAYVELSRLRSDDTAVYYCARDPYTSGFDYWGQGTLV TYSS [SEQ. ID. NO: 129] V_(H)12QVQLQESGPGLVKPSQTLSLTCTVSGGSIRSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCAREDTAMDYFDYWGQGT LVTVSS [SEQ. ID. NO: 141] V_(L)1DIVLTQSPDSLAVSLGERATINCKSSQSILYSSSNENFLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCQQYFSVFRTFGQGTRVEIK [SEQ. ID. NO: 3] V_(L)2EIVLTQSPGTLSLSPGDRATLSCRASQSVSSSYFAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYDRSPRTFGQGTKVEIK[SEQ. ID. NO: 15] V_(L)3 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYFAWYQQKPGQAPRLLIYGTSSRATGIPDRFSGSGSGTDFTLTV SRLEPEDFAVYYCQQYDRSPRTFGQGTKVEIK[SEQ. ID. NO: 27] V_(L)4 EIVLTQSPGTLSLSPGERATLSCRASQYISNTYLAWFQQKPGQAPRLLIYGAATRATGIPDRFSGSGSGTDFTFTI SRLEPEDFAVYYCQQYGSSPWTFGQGTTVEIK[SEQ. ID. NO: 39] V_(L)5 EVVLTQSPGTLSLSPGERATLSCRASQSVCSSYLAWYQQKPDQAPRLLISGASSRATGIPDRFSGSGSGTDFTLTI SSLEPEDFAVYYCQQYDRSPRTFGQGTKVEIK[SEQ. ID. NO: 51] V_(L)6 NFMLAQPHSVSESPGKTVTISCIRTSGSIASNYVQWYQSQRPGSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLDTISGLKTEDEAYYCQSCDISNVVFGGGTKLTVL [SEQ. ID. NO: 63] V_(L)7EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQVPRLLIYGTSSRATGIPDRFSGSGSGTDFTLTV SRLEPEDFAVYYCLQYDRSPRTFGQGTKVEIK[SEQ. ID. NO: 75] V_(L)8 EIVLTQSPGTLSLSLGERAILSCRASQSLSSIYLAWYQQKPGQAPGLLIYGASSRATGIPDRFSGSGSGTDFTLTI SSLEPEDFAVYYCQQYATSPWTFGQGTKVEVK[SEQ. ID. NO: 87] V_(L)9 DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGRGSGTDFTLTIS SLQPEDFATYYCQQSFSFPITFGPGTKVDIK[SEQ. ID. NO: 99] V_(L)10 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSGRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSFTGSSTWLFGGGTKLTVL [SEQ. ID. NO: 111] V_(L)11EIVLTQSPGTLSLSPGERATLSCRASPSVSSSYFAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYGWSPRTFGQGTKVEIK[SEQ. ID. NO: 123] V_(L)12 QSVLTQPPSASGTPGQRVTISCSGSRSHIGSNTVNWYQHLPGTAPKLLIYSNNHRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL [SEQ. ID. NO: 135]

Each of the heavy chain variable regions listed in TABLE 1 may becombined with any of the light chain variable regions shown in TABLE 1to form an antigen binding protein. Examples of such combinationsinclude V_(H)1 combined with any of V_(L)1, V_(L)2, V_(L)3, V_(L)4,V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11, and VL12, orV_(H)2 combined with any of V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11, and VL12, etc.

In some instances, the antigen binding protein includes at least oneheavy chain variable region and/or one light chain variable region fromthose listed in TABLE 1. In some instances, the antigen binding proteinincludes at least two different heavy chain variable regions and/orlight chain variable regions from those listed in TABLE 1. An example ofsuch an antigen binding protein comprises (a) one V_(H)1, and (b) one ofV_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, or V_(H)12.

Another example comprises (a) one V_(H)2, and (b) one of V_(H)1, V_(H)3,V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11, orV_(H)12. Again another example comprises (a) one V_(H)3, and (b) one ofV_(H)1, V_(H)2, V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10,V_(H)11, or V_(H)12 and the like.

Again another example of such an antigen binding protein comprises (a)one V_(L)1, and (b) one of V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6,V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11, or VL12. Again another example ofsuch an antigen binding protein comprises (a) one V_(L)2, and (b) one ofV_(L)1, V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10,VL11, or VL12. Again another example of such an antigen binding proteincomprises (a) one V_(L)3, and (b) one of V_(L)1, V_(L)2, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11, or VL12 and the like.

The various combinations of heavy chain variable regions may be combinedwith any of the various combinations of light chain variable regions.

In other instances, the antigen binding protein contains two identicallight chain variable regions and/or two identical heavy chain variableregions. As an example, the antigen binding protein may be an antibodyor immunologically functional fragment that includes two light chainvariable regions and two heavy chain variable regions in combinations ofpairs of light chain variable regions and pairs of heavy chain variableregions as listed in TABLE 1.

Some antibodies that are provided comprise a heavy chain variable domaincomprising a sequence of amino acids that differs from the sequence of aheavy chain variable domain selected from V_(H)1, V_(H)2, V_(H)3,V_(H)4, V_(H)5, V_(H)6, V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11, andV_(H)12 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15amino acid residues, wherein each such sequence difference isindependently either a deletion, insertion or substitution of one aminoacid. The heavy chain variable region in some antibodies comprises asequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%,97% or 99% sequence identity to the amino acid sequences of the heavychain variable region of V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6,V_(H)7, V_(H)8, V_(H)9, V_(H)10, V_(H)11, and V_(H)12.

Certain antibodies comprise a light chain variable domain comprising asequence of amino acids that differs from the sequence of a light chainvariable domain selected from V_(L)1, V_(L)2, V_(L)3, V_(L)4, V_(L)5,V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11, and VL12, at only 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues,wherein each such sequence difference is independently either adeletion, insertion or substitution of one amino acid. The light chainvariable region in some antibodies comprises a sequence of amino acidsthat has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequenceidentity to the amino acid sequences of the light chain variable regionof V_(L)2, V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9,V_(L)10, VL11, and VL12.

Still other antigen binding proteins, e.g., antibodies orimmunologically functional fragments include variant forms of a variantheavy chain and a variant light chain as just described.

2. CDRs

In a traditional antibody, the CDRs are embedded within a framework inthe heavy and light chain variable region where they constitute theregions responsible for antigen binding and recognition. Variabledomains of immunoglobulin chains of the same species generally exhibit asimilar overall structure, comprising relatively conserved frameworkregions (FR) joined by hypervariable regions, more often called“complementarity determining regions” or CDRs. A variable regioncomprises at least three heavy or light chain CDRs. The CDRs from thetwo chains of each heavy chain/light chain pair mentioned above aretypically aligned by the framework regions to form a structure thatbinds specifically with a specific epitope on the target protein (e.g.,GM-CSF). From N-terminal to C-terminal, naturally-occurring light andheavy chain variable regions both typically conform to the followingorder of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Anumbering system has been devised for assigning numbers to amino acidsthat occupy positions in each of these domains. Complementaritydetermining regions (CDRs) and framework regions (FR) of a givenantibody may be identified using this system. This numbering system isdefined in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed., US Dept. of Health and Human Services, PHS, NIH,NIH Publication No. 91-3242, 1991, or Chothia & Lesk, 1987, J. Mol.Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The CDRsprovided herein may not only be used to define the antigen bindingdomain of a traditional antibody structure, but may be embedded in avariety of other polypeptide structures, as described herein.

The antigen binding proteins disclosed herein are polypeptides intowhich one or more CDRs are grafted, inserted and/or joined. An antigenbinding protein can have 1, 2, 3, 4, 5 or 6 CDRs. However, it is alsocontemplated that an antigen binding protein can have more than sixCDRs. An antigen binding protein thus can have, for example, one heavychain CDR1 (“CDRH1”), and/or one heavy chain CDR2 (“CDRH2”), and/or oneheavy chain CDR3 (“CDRH3”), and/or one light chain CDR1 (“CDRL1”),and/or one light chain CDR2 (“CDRL2”), and/or one light chain CDR3(“CDRL3”). Some antigen binding proteins include both a CDRH3 and aCDRL3. Certain antigen binding proteins that are disclosed hereincomprise one or more amino acid sequences that are identical or havesubstantial sequence identity to the amino acid sequences of one or moreof the CDRs presented in TABLE 2 (CDRHs) and TABLE 3 (CDRLs).

TABLE 2 Exemplary CDRH Sequences SEQ ID NO: Amino Acid Sequence 10 GYYIH11 WINPNSGGTNSAQKFRG 12 EGGYSYGYFDY 22 GYYMH 23 WINPNSGGTNYAQKFKG 24DKWLDGFDY 35 WINPNSGGTNYAQKFQG 36 DRWLDAFDI 47 WINPNSGGTNYAQRFRG 48APYDWTFDY 59 WINPNSGGRNYAQKFQG 60 DRWLDAFEI 70 SYGIS 71WISAYNGNTDYAQKLQG 72 QRYYYSMDV 94 GYYMY 95 WINPNSGGTNYARKFQG 96RPWELPFDY 108 NGDYVFTYFDY 119 WINPNSGGTNYAQKFRG 120 FGYFGYYFDY 132DPYTSGFDY 142 SGGYYWS 143 YIYYSGSTYYNPSLKS 144 EDTAMDYFDY

TABLE 3 Exemplary CDRL Sequences SEQ ID NO: Amino Acid Sequence 4KSSQSILYSSSNENFLT 5 WASTRES 6 QQYFSVFRT 16 RASQSVSSSYFA 17 GASSRAT 18QQYDRSPRT 40 RASQYISNTYLA 41 GAATRAT 42 QQYGSSPWT 52 RASQSVCSSYLA 64IRTSGSIASNYVQ 65 EDDQRPS 66 QSCDISNVV 77 GTSSRAT 78 LQYDRSPRT 88RASQSLSSIYLA 90 QQYATSPWT 100 RASQSISNYLN 101 TASSLQS 102 QQSFSFPIT 112TGTSSDVGGYNYVS 113 EVSGRPS 114 SSFTGSSTWL 124 RASPSVSSSYFA 126 QQYGWSPRT136 SGSRSHIGSNTVN 137 SNNHRPS 138 AAWDDSLNGPV

In one aspect, the CDRs disclosed herein include consensus sequencesderived from groups of related monoclonal antibodies. As describedherein, a “consensus sequence” refers to amino acid sequences havingconserved amino acids common among a number of sequences and variableamino acids that vary within a given amino acid sequences. The CDRconsensus sequences provided include CDRs corresponding to each ofCDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

Consensus sequences were determined using standard phylogenic analysesof the CDRs corresponding to the V_(H) and V_(L) of anti-GM-CSFantibodies. The consensus sequences were determined by keeping the CDRscontiguous within the same sequence corresponding to a V_(H) or V_(L).

The CDRH1 consensus sequences include amino acid sequence consisting ofX₁X₂GX₃X₄FX₅X₆YX₇X₈X₉ (SEQ ID NO: 94) wherein X₁ is selected from thegroup consisting of G and no amino acid, X₂ is selected from the groupconsisting of G and no amino acid, X₃ is selected from the groupconsisting of Y and F, X₄ is selected from the group consisting of T andS, X₅ is selected from the group consisting of T, S and G, X₆ isselected from the group consisting of G and S, X₇ is selected from thegroup consisting of Y and G, X₈ is selected from the group consisting ofI and M, and X₉ is selected from the group consisting of H and S. In oneaspect, the CDRH1 consensus is SEQ ID NO: 94, X₁X₂GX₃X₄XFX₅X₆YX₇X₈X₉wherein X₁ is selected from the group consisting of G and no amino acid,X₂ is selected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of Y and F, X₄ is selected from thegroup consisting of T and S, X₅ is selected from the group consisting ofT, S and G, X₆ is selected from the group consisting of G and S, X₇ isselected from the group consisting of Y and G, X₈ is selected from thegroup consisting of I and M, and X₉ is selected from the groupconsisting of H and S.

The CDRH2 consensus sequence includes amino acid sequence consisting ofX₁X₂X₃X₄X₅X₆GX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅G (SEQ ID NO: 106) wherein X₁ isselected from the group consisting of W and no amino acid, X₂ isselected from the group consisting of I and Y, X₃ is selected from thegroup consisting of N, S and I, X₄ is selected from the group consistingof P, A and Y, X₅ is selected from the group consisting of N and Y, X₆is selected from the group consisting of S and N, X₇ is selected fromthe group consisting of G and N, X₈ is selected from the groupconsisting of T and R, X₉ is selected from the group consisting of N andD, X₁₀ is selected from the group consisting of Y and S, X₁₁ is selectedfrom the group consisting of A and N, X₁₂ is selected from the groupconsisting of Q and R, X₁₃ is selected from the group consisting of Kand R, X₁₄ is selected from the group consisting of F and L, and X₁₅ isselected from the group consisting of Q, K and R. In one aspect, theCDRH2 consensus sequences is WINPNSGGTNX1AX2X3FX4G, wherein X1 is Y orS, X2 is Q or R, X3 is K or R and X4 is R, K or Q (SEQ ID NO: 28).

The CDRH3 consensus sequence includes amino acid sequences selected fromthe group consisting of X₁X₂X₃X₄X₅X₆X₇X₈FDX₈ (SEQ ID NO: 83) wherein X₁is selected from the group consisting of E and no amino acid, X₂ isselected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of P, D and G, X₄ is selected fromthe group consisting of Y, W, R and K, X₅ is selected from the groupconsisting of S, W, F, and T, X₆ is selected from the group consistingof Y and L, X₇ is selected from the group consisting of D and G, X₈ isselected from the group consisting of Y, no amino acid, and A, and X₉ isselected from the group consisting of M, T, and V.

The CDRL1 consensus sequence includes an amino acid sequences selectedfrom the group consisting of KSSQSX₁LYSSX₂NX₃NX₄LX₅ (SEQ ID NO: 107)wherein X₁ is selected from the group consisting of V and I, X₂ isselected from the group consisting of S and N, X₃ is selected from thegroup consisting of E and K, X₄ is selected from the group consisting ofY and F, and X₅ is selected from the group consisting of T and A;RASX₁X₂X₃X₄X₅X₆YX₇X₈ (SEQ ID NO: 118) wherein X₁ is selected from thegroup consisting of Q and P, X₂ is selected from the group consisting ofS and Y, X₃ is selected from the group consisting of V, L and I, X₄ isselected from the group consisting of S and C, X₅ is selected from thegroup consisting of S and N, X₆ is selected from the group consisting ofS, I, T and no amino acid, X₇ is selected from the group consisting of Fand L, and X₈ is selected from the group consisting of A and N; andX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀NX₁₁VX₁₂ (SEQ ID NO: 125) wherein X₁ is selectedfrom the group consisting of I, S and T, X₂ is selected from the groupconsisting of R and G, X₃ is selected from the group consisting of T andS, X₄ is selected from the group consisting of R and S, X₅ is selectedfrom the group consisting of G and S, X₆ is selected from the groupconsisting of S, H and D, X₇ is selected from the group consisting of Iand V, X₈ is selected from the group consisting of A and G, X₉ isselected from the group consisting of no amino acid and G, X₁₀ isselected from the group consisting of S and Y, X₁₁ is selected from thegroup consisting of Y and T, and X₁₂ is selected from the groupconsisting of Q, N and S. In one aspect, the CDRL1 consensus sequence isRASQX1X2X3X4X5YX6A, wherein X1 is s or y, X2 is V, I or L, X3 is S or N,X4 is S or C, X4 is S, T, S or Y. X5 is F or L (SEQ ID NO: 30).

The CDRL2 consensus sequence includes amino acid sequences selected fromthe group consisting of X₁X₂X₃X₄X₅X₆X₇, (SEQ ID NO: 130) wherein X₁ isselected from the group consisting of G, T and W, X₂ is selected fromthe group consisting of T and A, X₃ is selected from the groupconsisting of S and A, X₄ is selected from the group consisting of S andT, X₅ is selected from the group consisting of R and L, X₆ is selectedfrom the group consisting of A, E and Q, and X₇ is selected from thegroup consisting of T and S; and X₁X₂X₃X₄RPS (SEQ ID NO: 131) wherein X₁is selected from the group consisting of E and S, X₂ is selected fromthe group consisting of D, V and N, X₃ is selected from the groupconsisting of D, S and N, and X₄ is selected from the group consistingof Q, G and H. Within one aspect, the CDRL2 consensus sequence isGX1SSRAT wherein X1 is a or T (SEQ ID NO: 34).

The CDRL3 consensus sequences include amino acid sequence selected fromthe group consisting of X₁QX₂X₃X₄X₅X₆X₇T (SEQ ID NO: 84) wherein X₁ isselected from the group consisting of Q and L, X₂ is selected from thegroup consisting of Y and s, X₃ is selected from the group consisting ofD, G and F, X₄ is selected from the group consisting of R, T, and S, X₅is selected from the group consisting of S and V, X₆ is selected fromthe group consisting of F and P, and X₇ is selected from the groupconsisting of R and W; and X₁X₂X₃X₄DSSNX₆X₆X₇ (SEQ ID NO: 89) wherein X₁is selected from the group consisting of S and A, X₂ is selected fromthe group consisting of S and A, X₃ is selected from the groupconsisting of W and F, X₄ is selected from the group consisting of D andT, X₅ is selected from the group consisting of G, W, and no amino acid,X₆ is selected from the group consisting of V, L, and P, and X₆ isselected from the group consisting of V and no amino acid. Within oneaspect the CDRL3 consensus sequence is QQX1X2X3X4X5X6T, wherein X1 is Yor S, X2 is F, G or A, X3 is S, T or W, X4 is V, S, F, X 5 is F or P, X6is R, W, or I (SEQ ID NO:46).

In another aspect, the CDRs provided are a (a) a CDRH selected from thegroup consisting of (i) a CDRH1 selected from the group consisting ofSEQ ID NO: 10, 22, 70 94 and 142; (ii) a CDRH2 selected from the groupconsisting of SEQ ID NO: 11, 23, 28, 35, 47, 59, 71, 95, 106, 119 and143; (iii) a CDRH3 selected from the group consisting of SEQ ID NO: 12,24, 36, 48, 60, 72, 83, 96, 108, 120, 132, and 144; and (iv) a CDRH of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than five, four, three, two, or oneamino acids; (B) a CDRL selected from the group consisting of (i) aCDRL1 selected from the group consisting of SEQ ID NO: 4, 16, 30, 40,52, 64, 88, 100, 107, 112, 118, 124, 125 and 136; (ii) a CDRL2 selectedfrom the group consisting of SEQ ID NO: 5, 17, 29, 34, 41, 65, 77, 101,113, 130, 131 and 137; (iii) a CDRL3 selected from the group consistingof SEQ ID NO: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114, 126, and 138;and (iv) a CDRL of (i), (ii) and (iii) that contains one or more aminoacid substitutions, deletions or insertions of no more than five, four,three, two, or one amino acids amino acids.

In yet another aspect, variant forms of the CDRs listed in TABLES 2 and3 have at least 80%, 85%, 90% or 95% sequence identity to a CDR sequencelisted in TABLE 2 and 3.

According to one aspect, provided is an isolated antigen-binding proteinthat binds GM-CSF comprising (A) one or more heavy chain complementarydetermining regions (CDRHs) selected from the group consisting of: (i) aCDRH1 selected from the group consisting of SEQ ID NO: 10, 22, 70 94 and142; (ii) a CDRH2 selected from the group consisting of SEQ ID NO: 11,23, 28, 35, 47, 59, 71, 95, 106, 119 and 143; (iii) a CDRH3 selectedfrom the group consisting of SEQ ID NO: 12, 24, 36, 48, 60, 72, 83, 96,108, 120, 132, and 144; and (iv) a CDRH of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than five, four, three, four, two or one amino acids; (B) oneor more light chain complementary determining regions (CDRLs) selectedfrom the group consisting of: (i) a CDRL1 selected from the groupconsisting of SEQ ID NO: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118,124, 125 and 136; (ii) a CDRL2 selected from the group consisting of SEQID NO: 5, 17, 29, 34, 41, 65, 77, 101, 113, 130, 131 and 137; (iii) aCDRL3 selected from the group consisting of SEQ ID NO: 6, 18, 42, 46,66, 78, 84, 89, 90, 102, 114, 126, and 138; and (iv) a CDRL of (i), (ii)and (iii) that contains one or more amino acid substitutions, deletionsor insertions of no more than five, four, three, four, two or one aminoacids; or (C) one or more heavy chain CDRHs of (A) and one or more lightchain CDRLs of (B).

In yet another embodiment, the isolated antigen-binding protein maycomprise (A) a CDRH selected from the group consisting of (i) a CDRH1selected from the group consisting of SEQ ID NO: 10, 22, 70 94 and 142;(ii) a CDRH2 selected from the group consisting of SEQ ID NO: 11, 23,28, 35, 47, 59, 71, 95, 106, 119 and 143; and (iii) a CDRH3 selectedfrom the group consisting of SEQ ID NO: 12, 24, 36, 48, 60, 72, 83, 96,108, 120, 132, and 144; (B) a CDRL selected from the group consisting of(i) a CDRL1 selected from the group consisting of SEQ ID NO: 4, 16, 30,40, 52, 64, 88, 100, 107, 112, 118, 124, 125 and 136; (ii) a CDRL2selected from the group consisting of SEQ ID NO: 5, 17, 29, 34, 41, 65,77, 101, 113, 130, 131 and 137; (iii) a CDRL3 selected from the groupconsisting of SEQ ID NO: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114,126, and 138; or (C) one or more heavy chain CDRHs of (A) and one ormore light chain CDRLs of (B).

In another embodiment, the variable heavy chain (VH) has at least 80%,85%, 90% or 95% sequence identity with an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 93,105, 117, 129, and 141, and/or the variable light chain (VL) has atleast 80%, 85%, 90% or 95% sequence identity with an amino acid sequenceselected from the group consisting of SEQ ID NO: 3, 15, 27, 39, 51, 63,75, 87, 99, 111, 123, and 135.

In a further aspect, there is a provision of an isolated antigen-bindingprotein that binds GM-CSF, the antigen-binding protein including A) aheavy chain complementary determining region (CDRH) selected from thegroup consisting of (i) a CDRH3 selected from the group consisting ofSEQ ID NOs: 12, 24, 36, 48, 60, 72, 83, 96, 108, 120, 132, and 144; (ii)a CDRH3 that differs in amino acid sequence from the CDRH3 of (i) by anamino acid addition, deletion or substitution of not more than two aminoacids; (iii) a CDRH3 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆X₇X₈FDX₉ (SEQ ID NO: 83) wherein X₁ isselected from the group consisting of E and no amino acid, X₂ isselected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of P, D and G, X₄ is selected fromthe group consisting of Y, W, R and K, X₅ is selected from the groupconsisting of S, W, F, and T, X₆ is selected from the group consistingof Y and L, X₇ is selected from the group consisting of D and G, X₈ isselected from the group consisting of Y, no amino acid, and A, and X₉ isselected from the group consisting of M, T, and V; and/or B) a lightchain complementary determining region (CDRL) selected from the groupconsisting of (i) a CDRL3 selected from the group consisting of SEQ IDNOs: 6, 18, 42, 46, 66, 78, 84, 89, 90, 102, 114, 126, and 138, (ii) aCDRL3 that differs in amino acid sequence from the CDRL3 of (i) by anamino acid addition, deletion or substitution of not more than two aminoacids; and iii) a CDRL3 amino acid sequence selected from the groupconsisting of X₁QX₂X₃X₄X₅X₆X₇T (SEQ ID NO: 84) wherein X1 is selectedfrom the group consisting of Q and L, X₂ is selected from the groupconsisting of Y and S, X₃ is selected from the group consisting of D, Gand F, X₄ is selected from the group consisting of R, T, and S, X₅ isselected from the group consisting of S and V, X₆ is selected from thegroup consisting of F and P, and X₇ is selected from the groupconsisting of R and W; and X₁X₂X₃X₄DSSNX₅X₆X₇ (SEQ ID NO: 89) wherein X₁is selected from the group consisting of S and A, X₂ is selected fromthe group consisting of S and A, X₃ is selected from the groupconsisting of W and F, X₄ is selected from the group consisting of D andT, X₅ is selected from the group consisting of G, W, and no amino acid,X6 is selected from the group consisting of V, L, and P, and X₆ isselected from the group consisting of V and no amino acid.

Within another embodiment, the antigen binding protein furthercomprising: A) a CDRH selected from the group consisting of: (i) a CDRH1selected from the group consisting of SEQ ID NOs: 10, 22, 70 94 and 142;(ii) a CDRH1 that differs in amino acid sequence from the CDRH1 of (i)by an amino acid addition, deletion or substitution of not more than twoamino acids; (iii) a CDRH1 amino acid sequence selected from the groupconsisting of X₁X₂GX₃X₄XFX₅X₆YX₇X₈X₉ (SEQ ID NO: 94) wherein X₁ isselected from the group consisting of G and no amino acid, X₂ isselected from the group consisting of G and no amino acid, X₃ isselected from the group consisting of Y and F, X₄ is selected from thegroup consisting of T and S, X₅ is selected from the group consisting ofT, S and G, X₆ is selected from the group consisting of G and S, X₇ isselected from the group consisting of Y and G, X₈ is selected from thegroup consisting of I and M, and X₉ is selected from the groupconsisting of H and S, or (iv) a CDRH2 selected from the groupconsisting of SEQ ID NOs: 11, 23, 28, 35, 47, 59, 71, 95, 106, 119 and143; (v) a CDRH2 that differs in amino acid sequence from the CDRH2 of(iv) by an amino acid addition, deletion or substitution of not morethan two amino acids; or (vi) a CDRH2 amino acid sequence consisting ofX₁X₂X₃X₄X₅X₆GX₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅G (SEQ ID NO: 106) wherein X₁ isselected from the group consisting of W and no amino acid, X₂ isselected from the group consisting of I and Y, X₃ is selected from thegroup consisting of N, S and I, X₄ is selected from the group consistingof P, A and Y, X₅ is selected from the group consisting of N and Y, X₆is selected from the group consisting of S and N, X₇ is selected fromthe group consisting of G and N, X₈ is selected from the groupconsisting of T and R, X₉ is selected from the group consisting of N andD, X₁₀ is selected from the group consisting of Y and S, X₁₁ is selectedfrom the group consisting of A and N, X₁₂ is selected from the groupconsisting of Q and R, X₁₃ is selected from the group consisting of Kand R, X₁₄ is selected from the group consisting of F and L, and X₁₅ isselected from the group consisting of Q, K and R; or B) a CDRL selectedfrom the group consisting of: (i) a CDRL1 selected from the groupconsisting of SEQ ID NOs: 4, 16, 30, 40, 52, 64, 88, 100, 107, 112, 118,124, 125 and 136; (ii) a CDRL1 that differs in amino acid sequence fromthe CDRL1 of (i) by an amino acid addition, deletion or substitution ofnot more than two amino acids; (iii) a CDRL1 amino acid sequenceselected from the group consisting of KSSQSX₁XLYSSX₂NX₃NX₄LX₅ (SEQ IDNO: 107) wherein X₁ is selected from the group consisting of V and I, X₂is selected from the group consisting of S and N, X₃ is selected fromthe group consisting of E and K, X₄ is selected from the groupconsisting of Y and F, and X₅ is selected from the group consisting of Tand A; RASX₁X₂X₃X₄X₅X₆YX₇X₈ (SEQ ID NO: 118) wherein X₁ is selected fromthe group consisting of Q and P, X₂ is selected from the groupconsisting of S and Y, X₃ is selected from the group consisting of V, Land I, X₄ is selected from the group consisting of S and C, X₅ isselected from the group consisting of S and N, X₆ is selected from thegroup consisting of S, I, T and no amino acid, X₇ is selected from thegroup consisting of F and L, and X₈ is selected from the groupconsisting of A and N; or X₁X₂X₃X₄X₅X₆YX₇X₈X₉X₁₀NX₁₁VX₁₂ (SEQ ID NO:125) wherein X₁ is selected from the group consisting of I, S and T, X₂is selected from the group consisting of R and G, X₃ is selected fromthe group consisting of T and S, X₄ is selected from the groupconsisting of R and S, X₅ is selected from the group consisting of G andS, X₆ is selected from the group consisting of S, H and D, X₇ isselected from the group consisting of I and V, X₈ is selected from thegroup consisting of A and G, X₉ is selected from the group consisting ofno amino acid and G, X₁₀ is selected from the group consisting of S andY, X₁₁ is selected from the group consisting of Y and T, and X₁₂ isselected from the group consisting of Q, N and S; or (iv) a CDRL2selected from the group consisting of SEQ ID NOs: 5, 17, 29, 34, 41, 65,77, 101, 113, 130, 131 and 137; (v) a CDRL2 that differs in amino acidsequence from the CDRL2 of (iv) by an amino acid addition, deletion orsubstitution of not more than two amino acids; or (vi) a CDRL2 aminoacid sequence selected from the group consisting of X₁X₂X₃X₄X₅X₆X₇ (SEQID NO: 130) wherein X1 is selected from the group consisting of G, T andW, X2 is selected from the group consisting of T and A, X3 is selectedfrom the group consisting of S and A, X4 is selected from the groupconsisting of S and T, X5 is selected from the group consisting of R andL, X6 is selected from the group consisting of A, E and Q, and X7 isselected from the group consisting of T and S; or X1X2X3X4RPS (SEQ IDNO: 131) wherein X1 is selected from the group consisting of E and S, X2is selected from the group consisting of D, V and N, X3 is selected fromthe group consisting of D, S and N, and X4 is selected from the groupconsisting of Q, G and H.

In one aspect, the isolated antigen-binding proteins provided herein canbe a monoclonal antibody, a polyclonal antibody, a recombinant antibody,a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof.

In another embodiment, the antibody fragment of the isolatedantigen-binding proteins provided herein can be a Fab fragment, a Fab′fragment, an F(ab′)₂ fragment, an Fv fragment, a diabody, or a singlechain antibody molecule.

In a further embodiment, the isolated antigen binding protein providedherein is a human antibody and can be of the IgG1-, IgG2- IgG3- orIgG4-type.

In yet another aspect, the isolated antigen-binding protein providedherein can be coupled to a labeling group and can compete for binding tothe extracellular portion of human GM-CSF with an antigen bindingprotein of one of the isolated antigen-binding proteins provided herein.In one embodiment, the isolated antigen binding protein provided hereincan reduce monocyte chemotaxis, inhibit monocyte migration into tumorsor inhibit accumulation of tumor associated macrophage in a tumor whenadministered to a patient.

As will be appreciated by those in the art, for any antigen bindingprotein with more than one CDR from the depicted sequences, anycombination of CDRs independently selected from the depicted sequencesis useful. Thus, antigen binding proteins with one, two, three, four,five or six of independently selected CDRs can be generated. However, aswill be appreciated by those in the art, specific embodiments generallyutilize combinations of CDRs that are non-repetitive, e.g., antigenbinding proteins are generally not made with two CDRH2 regions, etc.

Some of the antigen binding proteins provided are discussed in moredetail below.

Antigen Binding Proteins and Binding Epitopes

When an antigen binding protein is said to bind an epitope withinspecified residues, such as GM-CSF, or the extracellular domain ofGM-CSF, for example, what is meant is that the antigen binding proteinspecifically binds to a polypeptide consisting of the specified residues(e.g., a specified segment of GM-CSF). Such an antigen binding proteintypically does not contact every residue within GM-CSF, or theextracellular domain of GM-CSF. Nor does every single amino acidsubstitution or deletion within GM-CSF, or the extracellular domain ofGM-CSF, necessarily significantly affect binding affinity. Epitopespecificity of an antigen binding protein can be determined in varietyof ways. One approach, for example, involves testing a collection ofoverlapping peptides of about 15 amino acids spanning the sequence ofthe antigen and differing in increments of a small number of amino acids(e.g., three amino acids). The peptides are immobilized within the wellsof a microtiter dish. Immobilization can be effected by biotinylatingone terminus of the peptides. Optionally, different samples of the samepeptide can be biotinylated at the amino- and the carboxy-terminus andimmobilized in separate wells for purposes of comparison. This is usefulfor identifying end-specific antigen binding proteins. Optionally,additional peptides can be included terminating at a particular aminoacid of interest. This approach is useful for identifying end-specificantigen binding proteins to internal fragments of GM-CSF (or theextracellular domain of GM-CSF). An antigen binding protein orimmunologically functional fragment is screened for specific binding toeach of the various peptides. The epitope is defined as occurring with asegment of amino acids that is common to all peptides to which theantigen binding protein shows specific binding. Details regarding aspecific approach for defining an epitope are set forth in Example 13.

Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that competewith one the exemplified antibodies or functional fragments binding tothe epitope described above for specific binding to GM-CSF. Such antigenbinding proteins may also bind to the same epitope as one of the hereinexemplified antigen binding proteins, or an overlapping epitope. Antigenbinding proteins and fragments that compete with or bind to the sameepitope as the exemplified antigen binding proteins are expected to showsimilar functional properties. The exemplified antigen binding proteinsand fragments include those described above, including those with theheavy and light chains, variable region domains and CDRs included inTABLES 1, 2, and 3.

1. Monoclonal Antibodies

The antigen binding proteins that are provided include monoclonalantibodies that bind to GM-CSF. Monoclonal antibodies may be producedusing any technique known in the art, e.g., by immortalizing spleencells harvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with a GM-CSF immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds a GM-CSF polypeptide. Such hybridomacell lines, and anti-GM-CSF monoclonal antibodies produced by them, areaspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any technique known in the art. Hybridomas or mAbs may be furtherscreened to identify mAbs with particular properties, such as theability to block a Wnt induced activity. Examples of such screens areprovided in the examples below.

2. Chimeric and Humanized Antibodies

Chimeric and humanized antibodies based upon the foregoing sequences arealso provided. Monoclonal antibodies for use as therapeutic agents maybe modified in various ways prior to use. One example is a chimericantibody, which is an antibody composed of protein segments fromdifferent antibodies that are covalently joined to produce functionalimmunoglobulin light or heavy chains or immunologically functionalportions thereof. Generally, a portion of the heavy chain and/or lightchain is identical with or homologous to a corresponding sequence inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to a corresponding sequencein antibodies derived from another species or belonging to anotherantibody class or subclass. For methods relating to chimeric antibodies,see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., 1985,Proc. Natl. Acad. Sci. USA 81:6851-6855. CDR grafting is described, forexample, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089,and U.S. Pat. No. 5,530,101.

Generally, the goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended patient species ismaximized. One example is the “CDR-grafted” antibody, in which theantibody comprises one or more complementarity determining regions(CDRs) from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, the variable region or selected CDRs from arodent antibody often are grafted into a human antibody, replacing thenaturally-occurring variable regions or CDRs of the human antibody.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody of corresponding isotype. Humanization canbe performed, for example, using various methods by substituting atleast a portion of a rodent variable region for the correspondingregions of a human antibody (see, e.g., U.S. Pat. No. 5,585,089, and No.5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536),

In one aspect, the CDRs of the light and heavy chain variable regions ofthe antibodies provided herein (see, TABLE 2) are grafted to frameworkregions (FRs) from antibodies from the same, or a different,phylogenetic species. For example, the CDRs of the heavy and light chainvariable regions V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, V_(H)6, V_(H)7,V_(H)8, V_(H)9, V_(H)10, V_(H)11, and V_(H)12, and/or V_(L)1, V_(L)2,V_(L)3, V_(L)4, V_(L)5, V_(L)6, V_(L)7, V_(L)8, V_(L)9, V_(L)10, VL11,and VL12, can be grafted to consensus human FRs. To create consensushuman FRs, FRs from several human heavy chain or light chain amino acidsequences may be aligned to identify a consensus amino acid sequence. Inother embodiments, the FRs of a heavy chain or light chain disclosedherein are replaced with the FRs from a different heavy chain or lightchain. In one aspect, rare amino acids in the FRs of the heavy and lightchains of anti-GM-CSF antibody are not replaced, while the rest of theFR amino acids are replaced. A “rare amino acid” is a specific aminoacid that is in a position in which this particular amino acid is notusually found in an FR. Alternatively, the grafted variable regions fromthe one heavy or light chain may be used with a constant region that isdifferent from the constant region of that particular heavy or lightchain as disclosed herein. In other embodiments, the grafted variableregions are part of a single chain Fv antibody.

In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

3. Fully Human Antibodies

Fully human antibodies are also provided. Methods are available formaking fully human antibodies specific for a given antigen withoutexposing human beings to the antigen (“fully human antibodies”). Onespecific means provided for implementing the production of fully humanantibodies is the “humanization” of the mouse humoral immune system.Introduction of human immunoglobulin (Ig) loci into mice in which theendogenous Ig genes have been inactivated is one means of producingfully human monoclonal antibodies (mAbs) in mouse, an animal that can beimmunized with any desirable antigen. Using fully human antibodies canminimize the immunogenic and allergic responses that can sometimes becaused by administering mouse or mouse-derivatized mAbs to humans astherapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals(usually mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; andBruggermann et al., 1993, Year in Immunol. 7:33. In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, for example,WO96/33735 and WO94/02602. Additional methods relating to transgenicmice for making human antibodies are described in U.S. Pat. No.5,545,807; U.S. Pat. No. 6,713,610; U.S. Pat. No. 6,673,986; U.S. Pat.No. 6,162,963; U.S. Pat. No. 5,545,807; U.S. Pat. No. 6,300,129; U.S.Pat. No. 6,255,458; U.S. Pat. No. 5,877,397; U.S. Pat. No. 5,874,299 andU.S. Pat. No. 5,545,806; in PCT publications WO91/10741, WO90/04036, andin EP 546073B1 and EP 546073A1.

The transgenic mice described above, referred to herein as “HuMab” mice,contain a human immunoglobulin gene minilocus that encodes unrearrangedhuman heavy ([mu] and [gamma]) and [kappa] light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous [mu] and [kappa] chain loci (Lonberg et al., 1994, Nature368:856-859). Accordingly, the mice exhibit reduced expression of mouseIgM or [kappa] and in response to immunization, and the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG [kappa] monoclonalantibodies (Lonberg et al., supra.; Lonberg and Huszar, 1995, Intern.Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMab mice is described in detail inTaylor et al., 1992, Nucleic Acids Research 20:6287-6295; Chen et al.,1993, International Immunology 5:647-656; Tuaillon et al., 1994, J.Immunol. 152:2912-2920; Lonberg et al., 1994, Nature 368:856-859;Lonberg, 1994, Handbook of Exp. Pharmacology 113:49-101; Taylor et al.,1994, International Immunology 6:579-591; Lonberg and Huszar, 1995,Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. N.Y.Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology14:845-85. See, further U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; andU.S. Pat. No. 5,770,429; as well as U.S. Pat. No. 5,545,807; PCTPublication Nos. WO 93/1227; WO 92/22646; and WO 92/03918. Technologiesutilized for producing human antibodies in these transgenic mice aredisclosed also in PCT Publication No. WO 98/24893, and Mendez et al.,1997, Nature Genetics 15:146-156. For example, the HCo7 and HCo12transgenic mice strains can be used to generate anti-GM-CSF antibodies.

Using hybridoma technology, antigen-specific human mAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies may be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; andMarks et al., 1991, J. Mol. Biol. 222:581). Phage display techniquesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Publication No. WO 99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

4. Bispecific or Bifunctional Antigen Binding Proteins

The antigen binding proteins that are provided also include bispecificand bifunctional antibodies that include one or more CDRs or one or morevariable regions as described above. A bispecific or bifunctionalantibody in some instances is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol.79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

5. Various Other Forms

Some of the antigen binding proteins that are provided are variant formsof the antigen binding proteins disclosed above (e.g., those having thesequences listed in TABLES 1-4). For instance, some of the antigenbinding proteins have one or more conservative amino acid substitutionsin one or more of the heavy or light chains, variable regions or CDRslisted in TABLES 1-4.

Naturally-occurring amino acids may be divided into classes based oncommon side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues may be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, e.g.,Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certainamino acids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In some aspects, those which are within ±1are included, and in other aspects, those within ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, that is, with a biological propertyof the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in otherembodiments, those which are within ±1 are included, and in still otherembodiments, those within ±0.5 are included. In some instances, one mayalso identify epitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary conservative amino acid substitutions are set forth in TABLE4.

TABLE 4 Conservative Amino Acid Substitutions Original Exemplary ResidueSubstitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu MetLeu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile,Leu

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. Oneskilled in the art may identify suitable areas of the molecule that maybe changed without destroying activity by targeting regions not believedto be important for activity. The skilled artisan also will be able toidentify residues and portions of the molecules that are conserved amongsimilar polypeptides. In further embodiments, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure andamino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for GM-CSF neutralizing activity, (see examplesbelow) thus yielding information regarding which amino acids can bechanged and which must not be changed. In other words, based oninformation gathered from such routine experiments, one skilled in theart can readily determine the amino acid positions where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See, Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochem. 13:222-245; Chou et al., 1974,Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat. AreasMol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem. 47:251-276;and Chou et al., 1979, Biophys. J. 26:367-384. Moreover, computerprograms are currently available to assist with predicting secondarystructure. One method of predicting secondary structure is based uponhomology modeling. For example, two polypeptides or proteins that have asequence identity of greater than 30%, or similarity greater than 40%often have similar structural topologies. The recent growth of theprotein structural database (PDB) has provided enhanced predictabilityof secondary structure, including the potential number of folds within apolypeptide's or protein's structure. See, Holm et al., 1999, Nucl.Acid. Res. 27:244-247. It has been suggested (Brenner et al., 1997,Curr. Op. Struct. Biol. 7:369-376) that there are a limited number offolds in a given polypeptide or protein and that once a critical numberof structures have been resolved, structural prediction will becomedramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-387; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See, Holm, 1999, supra; and Brenner, 1997, supra).

In some embodiments, amino acid substitutions are made that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterligand or antigen binding affinities, and/or (4) confer or modify otherphysicochemical or functional properties on such polypeptides. Forexample, single or multiple amino acid substitutions (in certainembodiments, conservative amino acid substitutions) may be made in thenaturally-occurring sequence. Substitutions can be made in that portionof the antibody that lies outside the domain(s) forming intermolecularcontacts). In such embodiments, conservative amino acid substitutionscan be used that do not substantially change the structuralcharacteristics of the parent sequence (e.g., one or more replacementamino acids that do not disrupt the secondary structure thatcharacterizes the parent or native antigen binding protein). Examples ofart-recognized polypeptide secondary and tertiary structures aredescribed in Proteins, Structures and Molecular Principles (Creighton,Ed.), 1984, W. H. New York: Freeman and Company; Introduction to ProteinStructure (Branden and Tooze, eds.), 1991, New York: Garland Publishing;and Thornton et al., 1991, Nature 354:105.

Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues in the parent or native amino acidsequence are deleted from or substituted with another amino acid (e.g.,serine). Cysteine variants are useful, inter alia when antibodies mustbe refolded into a biologically active conformation. Cysteine variantsmay have fewer cysteine residues than the native antibody, and typicallyhave an even number to minimize interactions resulting from unpairedcysteines.

The heavy and light chains, variable regions domains and CDRs that aredisclosed can be used to prepare polypeptides that contain an antigenbinding region that can specifically bind to a GM-CSF polypeptide. Forexample, one or more of the CDRs listed in TABLES 3 and 4 can beincorporated into a molecule (e.g., a polypeptide) covalently ornoncovalently to make an immunoadhesion. An immunoadhesion mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDR(s) enable theimmunoadhesion to bind specifically to a particular antigen of interest(e.g., a GM-CSF polypeptide or epitope thereof).

Mimetics (e.g., “peptide mimetics” or “peptidomimetics”) based upon thevariable region domains and CDRs that are described herein are alsoprovided. These analogs can be peptides, non-peptides or combinations ofpeptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15:29;Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med.Chem. 30:1229. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Such compounds are often developedwith the aid of computerized molecular modeling. Generally,peptidomimetics are proteins that are structurally similar to anantibody displaying a desired biological activity, such as here theability to specifically bind GM-CSF, but have one or more peptidelinkages optionally replaced by a linkage selected from: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH—CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may be used incertain embodiments to generate more stable proteins. In addition,constrained peptides comprising a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61:387), forexample, by adding internal cysteine residues capable of formingintramolecular disulfide bridges which cyclize the peptide.

Derivatives of the antigen binding proteins that are described hereinare also provided. The derivatized antigen binding proteins can compriseany molecule or substance that imparts a desired property to theantibody or fragment, such as increased half-life in a particular use.The derivatized antigen binding protein can comprise, for example, adetectable (or labeling) moiety (e.g., a radioactive, colorimetric,antigenic or enzymatic molecule, a detectable bead (such as a magneticor electrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or Streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antigenbinding protein for a particular use (e.g., administration to a subject,such as a human subject, or other in vivo or in vitro uses). Examples ofmolecules that can be used to derivatize an antigen binding proteininclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antigen bindingproteins can be prepared using techniques well known in the art. In oneembodiment, the antigen binding protein is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyrrolidone), polyethyleneglycols, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates of GM-CSFantigen binding proteins with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of a GM-CSF antigenbinding protein. For example, the conjugated peptide may be aheterologous signal (or leader) polypeptide, e.g., the yeastalpha-factor leader, or a peptide such as an epitope tag. GM-CSF antigenbinding protein-containing fusion proteins can comprise peptides addedto facilitate purification or identification of the GM-CSF antigenbinding protein (e.g., poly-His). A GM-CSF antigen binding protein alsocan be linked to the FLAG peptide as described in Hopp et al., 1988,Bio/Technology 6:1204; and U.S. Pat. No. 5,011,912. The FLAG peptide ishighly antigenic and provides an epitope reversibly bound by a specificmonoclonal antibody (mAb), enabling rapid assay and facile purificationof expressed recombinant protein. Reagents useful for preparing fusionproteins in which the FLAG peptide is fused to a given polypeptide arecommercially available (Sigma, St. Louis, Mo.).

Oligomers that contain one or more GM-CSF antigen binding proteins maybe employed as GM-CSF antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more GM-CSF antigen bindingproteins are contemplated for use, with one example being a homodimer.Other oligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multipleGM-CSF-binding polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the GM-CSF antigenbinding proteins. Such peptides may be peptide linkers (spacers), orpeptides that have the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of GM-CSF antigen bindingproteins attached thereto, as described in more detail below.

In particular embodiments, the oligomers comprise from two to fourGM-CSF antigen binding proteins. The GM-CSF antigen binding proteinmoieties of the oligomer may be in any of the forms described above,e.g., variants or fragments. Preferably, the oligomers comprise GM-CSFantigen binding proteins that have GM-CSF binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a a GM-CSF antigen binding protein to the Fc region ofan antibody. The dimer can be made by, for example, inserting a genefusion encoding the fusion protein into an appropriate expressionvector, expressing the gene fusion in host cells transformed with therecombinant expression vector, and allowing the expressed fusion proteinto assemble much like antibody molecules, whereupon interchain disulfidebonds form between the Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT Publication No. WO93/10151 and U.S. Pat. Nos. 5,426,048 and 5,262,522, is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035, andin Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence ofthis mutein is identical to that of the native Fc sequence presented inPCT Publication No. WO 93/10151, except that amino acid 19 has beenchanged from Leu to Ala, amino acid 20 has been changed from Leu to Glu,and amino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a GM-CSF antigen binding protein such as disclosed herein maybe substituted for the variable portion of an antibody heavy and/orlight chain.

Alternatively, the oligomer is a fusion protein comprising multipleGM-CSF antigen binding proteins, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. No. 4,751,180 and U.S. Pat. No. 4,935,233.

Another method for preparing oligomeric GM-CSF antigen binding proteinderivatives involves use of a leucine zipper. Leucine zipper domains arepeptides that promote oligomerization of the proteins in which they arefound. Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT PublicationNo. WO 94/10308, and the leucine zipper derived from lung surfactantprotein D (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191.The use of a modified leucine zipper that allows for stabletrimerization of a heterologous protein fused thereto is described inFanslow et al., 1994, Semin. Immunol. 6:267-278. In one approach,recombinant fusion proteins comprising a GM-CSF antigen binding proteinfragment or derivative fused to a leucine zipper peptide are expressedin suitable host cells, and the soluble oligomeric GM-CSF antigenbinding protein fragments or derivatives that form are recovered fromthe culture supernatant.

Some antigen binding proteins that are provided have a binding affinity(K_(a)) for GM-CSF of at least 10⁴ or 10⁵/M×seconds measured, forinstance, as described in the examples below. Other antigen bindingproteins have a K_(a) of at least 10⁶, 10⁷, 10⁸ or 10⁹/M×seconds.Certain antigen binding proteins that are provided have a lowdisassociation rate. Some antibodies, for instance, have a K_(off) of1×10⁴ s⁻¹, 1×10⁻⁸ s⁻¹ or lower. In another embodiment, the K_(off) isthe same as an antibody having the following combinations of variableregion domains of TABLES 2 and 3.

Another aspect provides an antigen-binding protein having a half-life ofat least one day in vitro or in vivo (e.g., when administered to a humansubject). In one embodiment, the antigen binding protein has a half-lifeof at least three days. In another embodiment, the antibody or portionthereof has a half-life of four days or longer. In another embodiment,the antibody or portion thereof has a half-life of eight days or longer.In another embodiment, the antibody or antigen-binding portion thereofis derivatized or modified such that it has a longer half-life ascompared to the underivatized or unmodified antibody. In anotherembodiment, the antigen binding protein contains point mutations toincrease serum half life, such as described in PCT Publication No. WO00/09560.

6. Glycosylation

The antigen-binding protein may have a glycosylation pattern that isdifferent or altered from that found in the native species. As is knownin the art, glycosylation patterns can depend on both the sequence ofthe protein (e.g., the presence or absence of particular glycosylationamino acid residues, discussed below), or the host cell or organism inwhich the protein is produced. Particular expression systems arediscussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence may be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the target polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in PCT Publication No. WO87/05330, and in Aplin and Wriston, 1981, CRC Crit. Rev, Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Hence, aspects include glycosylation variants of the antigen bindingproteins wherein the number and/or type of glycosylation site(s) hasbeen altered compared to the amino acid sequences of the parentpolypeptide. In certain embodiments, antibody protein variants comprisea greater or a lesser number of N-linked glycosylation sites than thenative antibody. An N-linked glycosylation site is characterized by thesequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions that eliminate or alter this sequence willprevent addition of an N-linked carbohydrate chain present in the nativepolypeptide. For example, the glycosylation can be reduced by thedeletion of an Asn or by substituting the Asn with a different aminoacid. In other embodiments, one or more new N-linked sites are created.Antibodies typically have a N-linked glycosylation site in the Fcregion.

7. Labels And Effector Groups

In some embodiments, the antigen-binding comprises one or more labels.The term “labeling group” or “label” means any detectable label.Examples of suitable labeling groups include, but are not limited to,the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase),chemiluminescent groups, biotinyl groups, or predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used as is seen fit.

The term “effector group” means any group coupled to an antigen bindingprotein that acts as a cytotoxic agent. Examples for suitable effectorgroups are radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I). Other suitable groups include toxins,therapeutic groups, or chemotherapeutic groups. Examples of suitablegroups include calicheamicin, auristatins, geldanamycin and maytansine.In some embodiments, the effector group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookof Fluorescent Probes and Research Chemicals, Richard P. Haugland,Molecular Probes, 1992.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech, Mountain View, Calif., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec,Canada; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996,Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP,Clontech), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417),β galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:2603-2607) and Renilla (PCT Patent Application Nos. WO92/15673,WO95/07463, WO98/14605, WO98/26277, WO99/49019, U.S. Pat. No. 5,292,658,U.S. Pat. No. 5,418,155, U.S. Pat. No. 5,683,888, U.S. Pat. No.5,741,668, U.S. Pat. No. 5,777,079, U.S. Pat. No. 5,804,387, U.S. Pat.No. 5,874,304, U.S. Pat. No. 5,876,995, U.S. Pat. No. 5,925,558).

C. Nucleic Acids Encoding GM-CSF Antigen Binding Proteins

Nucleic acids that encode for the antigen binding proteins describedherein, or portions thereof, are also provided, including nucleic acidsencoding one or both chains of an antibody, or a fragment, derivative,mutein, or variant thereof, polynucleotides encoding heavy chainvariable regions or only CDRs, polynucleotides sufficient for use ashybridization probes, PCR primers or sequencing primers for identifying,analyzing, mutating or amplifying a polynucleotide encoding apolypeptide, anti-sense nucleic acids for inhibiting expression of apolynucleotide, and complementary sequences of the foregoing. Thenucleic acids can be any length. They can be, for example, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides inlength, including all values in between, and/or can comprise one or moreadditional sequences, for example, regulatory sequences, and/or be partof a larger nucleic acid, for example, a vector. The nucleic acids canbe single-stranded or double-stranded and can comprise RNA and/or DNAnucleotides and artificial variants thereof (e.g., peptide nucleicacids).

Nucleic acids encoding certain antigen binding proteins, or portionsthereof (e.g., full length antibody, heavy or light chain, variabledomain, or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) may be isolatedfrom B-cells of mice that have been immunized with GM-CSF or animmunogenic fragment thereof. The nucleic acid may be isolated byconventional procedures such as polymerase chain reaction (PCR). Phagedisplay is another example of a known technique whereby derivatives ofantibodies and other antigen binding proteins may be prepared. In oneapproach, polypeptides that are components of an antigen binding proteinof interest are expressed in any suitable recombinant expression system,and the expressed polypeptides are allowed to assemble to form antigenbinding protein molecules.

Due to the degeneracy of the genetic code, each of the polypeptidesequences listed in TABLES 1-4 or otherwise depicted herein are alsoencoded by a large number of other nucleic acid sequences besides thoseprovided. One of ordinary skill in the art will appreciate that thepresent application thus provides adequate written description andenablement for each degenerate nucleotide sequence encoding each antigenbinding protein.

An aspect further provides nucleic acids that hybridize to other nucleicacids under particular hybridization conditions. Methods for hybridizingnucleic acids are well-known in the art. See, e.g., Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Asdefined herein, a moderately stringent hybridization condition uses aprewashing solution containing 5× sodium chloride/sodium citrate (SSC),0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50%formamide, 6×SSC, and a hybridization temperature of 55° C. (or othersimilar hybridization solutions, such as one containing about 50%formamide, with a hybridization temperature of 42° C.), and washingconditions of 60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridizationcondition hybridizes in 6×SSC at 45° C., followed by one or more washesin 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art canmanipulate the hybridization and/or washing conditions to increase ordecrease the stringency of hybridization such that nucleic acidscomprising nucleotide sequences that are at least 65%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% identical to each other, including all valuesin between, typically remain hybridized to each other.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. and Current Protocols in Molecular Biology, 1995, Ausubelet al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), andcan be readily determined by those having ordinary skill in the artbased on, e.g., the length and/or base composition of the nucleic acid.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantibody or antibody derivative) that it encodes. Mutations can beintroduced using any technique known in the art. In one embodiment, oneor more particular amino acid residues are changed using, for example, asite-directed mutagenesis protocol. In another embodiment, one or morerandomly selected residues is changed using, for example, a randommutagenesis protocol. However it is made, a mutant polypeptide can beexpressed and screened for a desired property.

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. Alternatively, oneor more mutations can be introduced into a nucleic acid that selectivelychange the biological activity of a polypeptide that it encodes. Forexample, the mutation can quantitatively or qualitatively change thebiological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantibody.

Another aspect provides polynucleotides that are suitable for use asprimers or hybridization probes for the detection of nucleic acidsequences. A polynucleotide can comprise only a portion of a nucleicacid sequence encoding a full-length polypeptide, for example, afragment that can be used as a probe or primer or a fragment encoding anactive portion (e.g., a GM-CSF binding portion) of a polypeptide.

Probes based on the sequence of a nucleic acid can be used to detect thenucleic acid or similar nucleic acids, for example, transcripts encodinga polypeptide. The probe can comprise a label group, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used to identify a cell that expresses thepolypeptide.

Another aspect provides vectors comprising a nucleic acid encoding apolypeptide as described herein or a portion thereof (e.g., a fragmentcontaining one or more CDRs or one or more variable region domains).Examples of vectors include, but are not limited to, plasmids, viralvectors, non-episomal mammalian vectors and expression vectors, forexample, recombinant expression vectors. The recombinant expressionvectors can comprise a nucleic acid in a form suitable for expression ofthe nucleic acid in a host cell. The recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Regulatory sequences includethose that direct constitutive expression of a nucleotide sequence inmany types of host cells (e.g., SV40 early gene enhancer, Rous sarcomavirus promoter and cytomegalovirus promoter), those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences, see, Voss et al., 1986, TrendsBiochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, and thosethat direct inducible expression of a nucleotide sequence in response toparticular treatment or condition (e.g., the metallothionin promoter inmammalian cells and the tet-responsive and/or streptomycin responsivepromoter in both prokaryotic and eukaryotic systems (see, id.). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein.

Another aspect provides host cells into which a recombinant expressionvector has been introduced. A host cell can be any prokaryotic cell (forexample, E. coli) or eukaryotic cell (for example, yeast, insect, ormammalian cells (e.g., CHO cells)). Vector DNA can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. For stable transfection of mammalian cells, itis known that, depending upon the expression vector and transfectiontechnique used, only a small fraction of cells may integrate the foreignDNA into their genome. In order to identify and select these integrants,a gene that encodes a selectable marker (e.g., for resistance toantibiotics) is generally introduced into the host cells along with thegene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate.Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die), amongother methods.

D. Preparing of Antigen Binding Proteins

Fully human antibodies may be prepared as described above by immunizingtransgenic animals containing human immunoglobulin loci or by selectinga phage display library that is expressing a repertoire of humanantibodies.

The monoclonal antibodies (mAbs) can be produced by a variety oftechniques, including conventional monoclonal antibody methodology,e.g., the standard somatic cell hybridization technique of Kohler andMilstein, 1975, Nature 256:495. Alternatively, other techniques forproducing monoclonal antibodies can be employed, for example, the viralor oncogenic transformation of B-lymphocytes. One suitable animal systemfor preparing hybridomas is the murine system, which is a very wellestablished procedure. Immunization protocols and techniques forisolation of immunized splenocytes for fusion are known in the art. Forsuch procedures, B cells from immunized mice are fused with a suitableimmortalized fusion partner, such as a murine myeloma cell line. Ifdesired, rats or other mammals besides can be immunized instead of miceand B cells from such animals can be fused with the murine myeloma cellline to form hybridomas. Alternatively, a myeloma cell line from asource other than mouse may be used. Fusion procedures for makinghybridomas also are well known.

The single chain antibodies that are provided may be formed by linkingheavy and light chain variable domain (Fv region) fragments via an aminoacid bridge (short peptide linker), resulting in a single polypeptidechain. Such single-chain Fvs (scFvs) may be prepared by fusing DNAencoding a peptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:5879; Ward et al., 1989, Nature 334:544, de Graafet al., 2002, Methods Mol. Biol. 178:379-387. Single chain antibodiesderived from antibodies provided herein include, but are not limited toscFvs comprising the variable domain combinations of the heavy and lightchain variable regions depicted in TABLE 1, or combinations of light andheavy chain variable domains having grafted into any of the CDRsdepicted in TABLES 2 and 3.

Antibodies provided herein that are of one subclass can be changed toantibodies from a different subclass using subclass switching methods.Thus, IgG antibodies may be derived from an IgM antibody, for example,and vice versa. Such techniques allow the preparation of new antibodiesthat possess the antigen binding properties of a given antibody (theparent antibody), but also exhibit biological properties associated withan antibody isotype or subclass different from that of the parentantibody. Recombinant DNA techniques may be employed. Cloned DNAencoding particular antibody polypeptides may be employed in suchprocedures, e.g., DNA encoding the constant domain of an antibody of thedesired isotype. See, e.g., Lantto et al., 2002, Methods Mol. Biol.178:303-316. Accordingly, the antibodies that are provided include thosecomprising, for example, the variable domain combinations describedabove having a desired isotype (for example, IgA, IgG1, IgG2, IgG3,IgG4, IgE, and IgD) as well as Fab or F(ab′)₂ fragments thereof.Moreover, if an IgG4 is desired, it may also be desired to introduce apoint mutation (CPSCP->CPPCP) in the hinge region as described in Bloomet al., 1997, Protein Science 6:407) to alleviate a tendency to formintra-H chain disulfide bonds that can lead to heterogeneity in the IgG4antibodies.

Moreover, techniques for deriving antibodies having different properties(i.e., varying affinities for the antigen to which they bind) are alsoknown. One such technique, referred to as chain shuffling, involvesdisplaying immunoglobulin variable domain gene repertoires on thesurface of filamentous bacteriophage, often referred to as phagedisplay. Chain shuffling has been used to prepare high affinityantibodies to the hapten 2-phenyloxazol-5-one, as described by Marks etal., 1992, BioTechnology 10:779.

Conservative modifications may be made to the heavy and light chainvariable regions described in TABLE 1, or the CDRs described in TABLE 2and 3 (and corresponding modifications to the encoding nucleic acids) toproduce a GM-CSF antigen binding protein having functional andbiochemical characteristics. Methods for achieving such modificationsare described above.

GM-CSF antigen binding proteins may be further modified in various ways.For example, if they are to be used for therapeutic purposes, they maybe conjugated with polyethylene glycol (pegylated) to prolong the serumhalf-life or to enhance protein delivery. Alternatively, the V region ofthe subject antibodies or fragments thereof may be fused with the Fcregion of a different antibody molecule. The Fc region used for thispurpose may be modified so that it does not bind complement, thusreducing the likelihood of inducing cell lysis in the patient when thefusion protein is used as a therapeutic agent. In addition, the subjectantibodies or functional fragments thereof may be conjugated with humanserum albumin to enhance the serum half-life of the antibody or fragmentthereof. Another useful fusion partner for the inventive antibodies orfragments thereof is transthyretin (TTR). TTR has the capacity to form atetramer, thus an antibody-TTR fusion protein can form a multivalentantibody which may increase its binding avidity.

Alternatively, substantial modifications in the functional and/orbiochemical characteristics of the antigen binding proteins describedherein may be achieved by creating substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulkiness of the side chain. A “conservativeamino acid substitution” may involve a substitution of a native aminoacid residue with a normative residue that has little or no effect onthe polarity or charge of the amino acid residue at that position. See,TABLE 5. Furthermore, any native residue in the polypeptide may also besubstituted with alanine, as has been previously described for alaninescanning mutagenesis.

Amino acid substitutions (whether conservative or non-conservative) ofthe subject antibodies can be implemented by those skilled in the art byapplying routine techniques. Amino acid substitutions can be used toidentify important residues of the antibodies provided herein, or toincrease or decrease the affinity of these antibodies for human GM-CSFor for modifying the binding affinity of other antigen-binding proteinsdescribed herein.

E. Methods of Expressing Antigen Binding Proteins

Expression systems and constructs in the form of plasmids, expressionvectors, transcription or expression cassettes that comprise at leastone polynucleotide as described above are also provided herein, as wellhost cells comprising such expression systems or constructs.

The antigen binding proteins provided herein may be prepared by any of anumber of conventional techniques. For example, GM-CSF antigen bindingproteins may be produced by recombinant expression systems, using anytechnique known in the art. See, e.g., Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.)Plenum Press, New York (1980); and Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1988).

Antigen binding proteins can be expressed in hybridoma cell lines (e.g.,in particular antibodies may be expressed in hybridomas) or in celllines other than hybridomas. Expression constructs encoding theantibodies can be used to transform a mammalian, insect or microbialhost cell. Transformation can be performed using any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus or bacteriophage and transducinga host cell with the construct by transfection procedures known in theart, as exemplified by U.S. Pat. No. 4,399,216; U.S. Pat. No. 4,912,040;U.S. Pat. No. 4,740,461; U.S. Pat. No. 4,959,455. The optimaltransformation procedure used will depend upon which type of host cellis being transformed. Methods for introduction of heterologouspolynucleotides into mammalian cells are well known in the art andinclude, but are not limited to, dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, mixing nucleic acid with positively-charged lipids, anddirect microinjection of the DNA into nuclei.

Recombinant expression constructs typically comprise a polynucleotideencoding a polypeptide comprising one or more of the following: one ormore CDRs provided herein; a light chain constant region; a light chainvariable region; a heavy chain constant region (e.g., C_(H)1, C_(H)2and/or C_(H)3); and/or another scaffold portion of a GM-CSF antigenbinding protein. These nucleic acid sequences are inserted into anappropriate expression vector using standard ligation techniques. In oneembodiment, the heavy or light chain constant region is appended to theC-terminus of the anti-GM-CSF-specific heavy or light chain variableregion and is ligated into an expression vector. The vector is typicallyselected to be functional in the particular host cell employed (i.e.,the vector is compatible with the host cell machinery, permittingamplification and/or expression of the gene can occur). In someembodiments, vectors are used that employ protein-fragmentcomplementation assays using protein reporters, such as dihydrofolatereductase (see, for example, U.S. Pat. No. 6,270,964). Suitableexpression vectors can be purchased, for example, from Invitrogen LifeTechnologies (Carlsbad, Calif.) or BD Biosciences (San Jose, Calif.).Other useful vectors for cloning and expressing the antibodies andfragments include those described in Bianchi and McGrew, 2003, Biotech.Biotechnol. Bioeng. 84:439-44. Additional suitable expression vectorsare discussed, for example, in Methods Enzymol., vol. 185 (D. V.Goeddel, ed.), 1990, New York: Academic Press.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the GM-CSFantigen binding protein coding sequence; the oligonucleotide sequenceencodes polyHis (such as hexaHis), or another “tag” such as FLAG®, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the GM-CSF antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified GM-CSF antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors may be obtained by any ofseveral methods well known in the art. Typically, flanking sequencesuseful herein will have been previously identified by mapping and/or byrestriction endonuclease digestion and can thus be isolated from theproper tissue source using the appropriate restriction endonucleases. Insome cases, the full nucleotide sequence of a flanking sequence may beknown. Here, the flanking sequence may be synthesized using the methodsdescribed herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Qiagen, Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent toone of ordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein that binds to GM-CSF polypeptide. As a result,increased quantities of a polypeptide such as an antigen binding proteinare synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orpro-sequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein), one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning will typically contain a promoter that isrecognized by the host organism and operably linked to the moleculeencoding GM-CSF antigen binding protein. Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, uniformly transcribe a geneto which they are operably linked, that is, with little or no controlover gene expression. A large number of promoters, recognized by avariety of potential host cells, are well known. A suitable promoter isoperably linked to the DNA encoding heavy chain or light chaincomprising a GM-CSF antigen binding protein by removing the promoterfrom the source DNA by restriction enzyme digestion and inserting thedesired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40(SV40). Other suitable mammalian promoters include heterologousmammalian promoters, for example, heat-shock promoters and the actinpromoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene (Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising ahuman GM-CSF antigen binding protein by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 bp in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The expression vectors that are provided may be constructed from astarting vector such as a commercially available vector. Such vectorsmay or may not contain all of the desired flanking sequences. Where oneor more of the flanking sequences described herein are not alreadypresent in the vector, they may be individually obtained and ligatedinto the vector. Methods used for obtaining each of the flankingsequences are well known to one skilled in the art.

After the vector has been constructed and a polynucleotide encodinglight chain, a heavy chain, or a light chain and a heavy chaincomprising a GM-CSF antigen binding sequence has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an antigen-binding proteininto a selected host cell may be accomplished by well known methodsincluding transfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anantigen binding protein that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines may be selected throughdetermining which cell lines have high expression levels andconstitutively produce antigen binding proteins with GM-CSF bindingproperties. In another embodiment, a cell line from the B cell lineagethat does not make its own antibody but has a capacity to make andsecrete a heterologous antibody can be selected.

F. Use of Human GM-CSF Antigen Binding Proteins for Diagnostic andTherapeutic Purposes

Antigen binding proteins are useful for detecting GM-CSF in biologicalsamples and identification of cells or tissues that produce GM-CSF.Antigen binding proteins that specifically bind to GM-CSF may be used intreatment of diseases related to GM-CSF in a patient in need thereof.For one, the GM-CSF antigen binding proteins can be used in diagnosticassays, e.g., binding assays to detect and/or quantify GM-CSF expressedin a tissue or cell. In addition, GM-CSF antigen binding proteins can beused to inhibit GM-CSF from forming a complex with its receptor, therebymodulating the biological activity of GM-CSF in a cell or tissue.Antigen binding proteins that bind to GM-CSF thus may modulate and/orblock interaction with other binding compounds and as such may havetherapeutic use in ameliorating diseases related to GM-CSF.

1. Indications

The present invention also relates to the use of GM-CSF inhibitors (asdisclosed), such as GM-CSF antibodies, in the manufacture of amedicament for the prevention or therapeutic treatment of each medicaldisorder disclosed herein. The GM-CSF inhibitors are useful to treat avariety of conditions in which excess GM-CSF plays a role incontributing to the underlying disease or disorder or otherwisecontributes to a negative symptom.

Embodiments of the present invention include methods for using thedisclosed GM-CSF inhibitors, in particular GM-CSF antibodies,compositions or combination therapies to treat or prevent a variety ofrheumatic disorders. These include adult and juvenile rheumatoidarthritis; scleroderma; systemic lupus erythematosus; gout;osteoarthritis; polymyalgia rheumatica; seronegativespondylarthropathies, including ankylosing spondylitis, and Reiter'sdisease. The subject GM-CSF inhibitors, compositions and combinationtherapies are used also to treat psoriatic arthritis and chronic Lymearthritis. Also treatable or preventable with these compounds,compositions and combination therapies are Still's disease and uveitisassociated with rheumatoid arthritis. In addition, the compounds,compositions and combination therapies of the invention are used intreating disorders resulting in inflammation of the voluntary muscle andother muscles, including dermatomyositis, inclusion body myositis,polymyositis, and lymphangioleimyomatosis.

The subject invention provides GM-CSF inhibitors, e.g. GM-CSFantibodies, compositions and combination therapies (e.g. GM-CSFinhibitor and a TNF inhibitor such as ENBREL® (etanercept) or otheractive agents) for the treatment of non-arthritic medical conditions ofthe bones and joints. This encompasses osteoclast disorders that lead tobone loss, such as but not limited to osteoporosis, includingpost-menopausal osteoporosis, osteoarthritis, periodontitis resulting intooth loosening or loss, and prosthesis loosening after jointreplacement (generally associated with an inflammatory response to weardebris). This latter condition also is called “orthopedic implantosteolysis.” Another condition treatable with the compounds,compositions and combination therapies of the invention is temporalmandibular joint dysfunction (TMJ).

Various other medical disorders treatable with the disclosed GM-CSFinhibitor compositions and combination therapies include; multiplesclerosis; Behcet's syndrome; Sjogren's syndrome; autoimmune hemolyticanemia; beta thalassemia; amyotrophic lateral sclerosis (Lou Gehrig'sDisease); Parkinson's disease; and tenosynovitis of unknown cause, aswell as various autoimmune disorders or diseases associated withhereditary deficiencies, including x-linked mental retardation.

Also provided are methods for using GM-CSF inhibitors, compositions orcombination therapies to treat various disorders of the endocrinesystem. For example, GM-CSF inhibitor compositions or other GM-CSFinhibitor compositions, with or without TNF inhibitors (e.g., ENBREL) orother active agents described above, are suitable for use to treatjuvenile onset diabetes (includes autoimmune diabetes mellitus andinsulin-dependent types of diabetes) and also to treat maturity onsetdiabetes (includes non-insulin dependent and obesity-mediated diabetes).In addition, the subject compounds, compositions and combinationtherapies are used to treat secondary conditions associated withdiabetes, such as diabetic retinopathy, kidney transplant rejection indiabetic patients, obesity-mediated insulin resistance, and renalfailure, which itself may be associated with proteinurea andhypertension. Other endocrine disorders also are treatable with thesecompounds, compositions or combination therapies, including polycysticovarian disease, X-linked adrenoleukodystrophy, hypothyroidism andthyroiditis, including Hashimoto's thyroiditis (i.e., autoimmunethyroiditis). Further, GM-CSF inhibitors, including GM-CSF inhibitor,alone or in combination with other cytokines, including TNF inhibitorssuch as ENBREL, are useful in treating or preventing medical conditionsassociated with thyroid cell dysfunction, including euthyroid sicksyndrome.

Conditions of the gastrointestinal system are treatable or preventablewith GM-CSF inhibitors, compositions or combination therapies, includingcoeliac disease. For example, GM-CSF inhibitor compositions, with orwithout TNF inhibitors (e.g., ENBREL) or other active agents describedabove are suitable for treating or preventing coeliac disease. Inaddition, the compounds, compositions and combination therapies of theinvention are suitable for treating or preventing Crohn's disease;ulcerative colitis; idiopathic gastroparesis; pancreatitis, includingchronic pancreatitis; acute pancreatitis, inflammatory bowel disease andulcers, including gastric and duodenal ulcers.

Included also are methods for using the subject GM-CSF inhibitors,compositions or combination therapies for treating disorders of thegenitourinary system. For example, GM-CSF inhibitor compositions, aloneor in combination with IL-1 (e.g., Kineret® (anakinra)) or TNFinhibitors (e.g., ENBREL) or other active agents described above aresuitable for treating or preventing glomerulonephritis, includingautoimmune glomerulonephritis, glomerulonephritis due to exposure totoxins or glomerulonephritis secondary to infections with haemolyticstreptococci or other infectious agents. Also treatable with thecompounds, compositions and combination therapies of the invention areuremic syndrome and its clinical complications (for example, renalfailure, anemia, and hypertrophic cardiomyopathy), including uremicsyndrome associated with exposure to environmental toxins, drugs orother causes. GM-CSF inhibitors, particularly GM-CSF antibodies, aloneor in combination with TNF inhibitors, particularly ENBREL, are usefulin treating and preventing complications that arise from inflammation ofthe gallbladder wall that leads to alteration in absorptive function.Included in such complications are cholelithiasis (gallstones) andcholiedocholithiasis (bile duct stones) and the recurrence ofcholelithiasis and choliedocholithiasis. Further conditions treatablewith the compounds, compositions and combination therapies of theinvention are complications of hemodialysis; prostate conditions,including benign prostatic hypertrophy, nonbacterial prostatitis andchronic prostatitis; and complications of hemodialysis.

Also provided herein are methods for using GM-CSF inhibitors,compositions or combination therapies to treat various hematologic andoncologic disorders. For example, GM-CSF inhibitor, alone or incombination with an GM-CSF inhibitor, TNF inhibitor (e.g., ENBREL) orother active agents as described above, may be used to treat symptomsassociated with various forms of cancer, including acute myelogenousleukemia, chronic myelogenous leukemia, Epstein-Barr virus-positivenasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell,cervical and ovarian cancers, lung cancer (SCLC and NSCLC), includingcancer-associated cachexia, fatigue, asthenia, paraneoplastic syndromeof cachexia and hypercalcemia. Additional diseases treatable with thesubject GM-CSF inhibitors, compositions or combination therapies aresolid tumors, including sarcoma, osteosarcoma, and carcinoma, such asadenocarcinoma (for example, breast cancer) and squamous cell carcinoma.In addition, the subject compounds, compositions or combinationtherapies are useful for treating esophogeal cancer, gastric cancer,gall bladder carcinoma, leukemia, including acute myelogenous leukemia,chronic myelogenous leukemia, myeloid leukemia, chronic or acutelymphoblastic leukemia and hairy cell leukemia. Other malignancies withinvasive metastatic potential, including multiple myeloma, can betreated with the subject compounds, compositions and combinationtherapies, and particularly combination therapies that include GM-CSFinhibitor and soluble TNF receptor (e.g., ENBREL). In addition, thedisclosed GM-CSF inhibitors, compositions and combination therapies canbe used to treat anemias and hematologic disorders, including chronicidiopathic neutropenia, anemia of chronic disease, aplastic anemia,including Fanconi's aplastic anemia; idiopathic thrombocytopenic purpura(ITP); thrombotic thrombocytopenic purpura, myelodysplastic syndromes(including refractory anemia, refractory anemia with ringedsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation); myelofibrosis/myeloid metaplasia;and sickle cell vasocclusive crisis.

Various lymphoproliferative disorders also are treatable with thedisclosed GM-CSF inhibitors, compositions or combination therapies.GM-CSF inhibitor, alone or in combination with a TNF inhibitor, such asENBREL, or other active agents are useful for treating or preventingautoimmune lymphoproliferative syndrome (ALPS), chronic lymphoblasticleukemia, hairy cell leukemia, chronic lymphatic leukemia, peripheralT-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma,follicular lymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive Tcell lymphoma, histiocytic lymphoma, Hodgkin's disease, diffuseaggressive lymphoma, acute lymphatic leukemias, T gammalymphoproliferative disease, cutaneous B cell lymphoma, cutaneous T celllymphoma (i.e., mycosis fungoides) and Sezary syndrome.

In addition, the subject GM-CSF inhibitors, compositions and combinationtherapies are used to treat hereditary conditions. In particular, GM-CSFinhibitor, alone or in combination with a TNF inhibitor such as ENBREL,is useful to treat diseases such as Gaucher's disease, Huntington'sdisease, linear IgA disease, and muscular dystrophy.

Other conditions treatable or preventable by the disclosed GM-CSFinhibitors, compositions and combination therapies include thoseresulting from injuries to the head or spinal cord including subduralhematoma due to trauma to the head. For example, GM-CSF inhibitor, aloneor in combination with a TNF inhibitor such as ENBREL are useful intreating head injuries and spinal chord injuries. In connection withthis therapy, the compositions and combinations described are suitablefor preventing cranial neurologic damage and preventing and treatingcervicogenic headache. The compositions and combinations described arefurther suitable for treating neurological side effects associated withbrain irradiation.

The disclosed GM-CSF inhibitors, compositions and combination therapiesare further used to treat conditions of the liver. For example GM-CSFinhibitor, alone or in combination with a TNF inhibitor such as ENBRELor other active agents, can be used to treat hepatitis, including acutealcoholic hepatitis, acute drug-induced or viral hepatitis, hepatitis A,B and C, sclerosing cholangitis and inflammation of the liver due tounknown causes. In connection with liver inflammation, GM-CSF inhibitorsare further useful in treating hepatic sinusoid epithelium.

In addition, the disclosed GM-CSF inhibitors, compositions andcombination therapies are used to treat various disorders that involvehearing loss and that are associated with abnormal IL-1 expression. Forexample, GM-CSF inhibitor, alone or in combination with TNF inhibitors,can be used to treat or prevent cochlear nerve-associated hearing lossthat is thought to result from an autoimmune process, i.e., autoimmunehearing loss. This condition currently is treated with steroids,methotrexate and/or cyclophosphamide. Also treatable or preventable withthe disclosed GM-CSF inhibitors, compositions and combination therapiesis Meniere's syndrome and cholesteatoma, a middle ear disorder oftenassociated with hearing loss.

Disorders associated with transplantation also are treatable orpreventable with the disclosed GM-CSF inhibitors compositions orcombination therapies. Such disorders include graft-versus-host disease,and complications resulting from solid organ transplantation, such asheart, liver, skin, kidney, lung (lung transplant airway obliteration)or other transplants, including bone marrow transplants.

Ocular disorders also are treatable or preventable with the disclosedGM-CSF inhibitors, especially GM-CSF antibodies, compositions orcombination therapies, including rhegmatogenous retinal detachment, andinflammatory eye disease, including inflammatory eye disease associatedwith smoking and macular degeneration.

GM-CSF inhibitor compositions and combination therapies also are usefulfor treating disorders that affect the female reproductive system.Examples include, but are not limited to, multiple implantfailure/infertility; fetal loss syndrome or IV embryo loss (spontaneousabortion); preeclamptic pregnancies or eclampsia; endometriosis, chroniccervicitis, and pre-term labor.

In addition, the disclosed GM-CSF inhibitor compositions and combinationtherapies are useful for treating or preventing sciatica, symptoms ofaging, severe drug reactions (for example, 11-2 toxicity orbleomycin-induced pneumopathy and fibrosis), or to suppress theinflammatory response prior, during or after the transfusion ofallogeneic red blood cells in cardiac or other surgery, or in treating atraumatic injury to a limb or joint, such as traumatic knee injury.

The disclosed GM-CSF inhibitor compositions and combination therapiesare useful for treating central nervous system (CNS) injuries, includingthe effects of neurotoxic neurotransmitters discharged during excitationof inflammation in the central nervous system and to inhibit or preventthe development of glial scars at sites of central nervous systeminjury. In connection with central nervous system medical conditions,GM-CSF inhibitors are useful in treating temporal lobe epilepsy. Inconnection with epilepsy and the treatment of seizures, reducing theseverity and number of recurring seizures, and reducing the severity ofthe deleterious effects of seizures. GM-CSF inhibitors alone or incombination with agents described herein are useful for reducingneuronal loss, neuronal degeneration, and gliosis associated withseizures.

Furthermore, the disclosed GM-CSF inhibitor compositions and combinationtherapies are useful for treating critical illness polyneuropathy andmyopathy (CIPNM) acute polyneuropathy; anorexia nervosa; Bell's palsy;chronic fatigue syndrome; transmissible dementia, includingCreutzfeld-Jacob disease; demyelinating neuropathy; Guillain-Barresyndrome; vertebral disc disease; Gulf war syndrome; chronicinflammatory demyelinating polyneuropathy, myasthenia gravis; silentcerebral ischemia; sleep disorders, including narcolepsy and sleepapnea; chronic neuronal degeneration; and stroke, including cerebralischemic diseases.

Other diseases and medical conditions that may be treated or preventedby administering a GM-CSF inhibitor alone or in combination with aherein described active agents include anorexia and/or anorexicconditions, peritonitis, endotoxemia and septic shock, granulomaformation, heat stroke, Churg-Strauss syndrome, chronic inflammationfollowing acute infections such as tuberculosis and leprosy, systemicsclerosis and hypertrophic scarring. In addition to GM-CSF inhibitors incombination with IL-1 inhibitors, TNF inhibitors, IFN-alpha, -beta or-gamma and/or IL-4 inhibitors are suitable for treating hypertrophicscarring.

The GM-CSF inhibitors disclosed herein are useful for reducing thetoxicity associated with antibody therapies, chemotherapy, radiationtherapy and the effects of other apoptosis inducing agents, e.g. TRAILand TRADE.

Provided herein are methods of treating or preventing psoriatic lesionsthat involve administering to a human patient a therapeuticallyeffective amount of a GM-CSF inhibitor. The treatment is effectiveagainst psoriatic lesions that occur in patients who have ordinarypsoriasis or psoriatic arthritis.

Conditions effectively treated by a GM-CSF inhibitor play a role in theinflammatory response. Lung disorders include asthma, chronicobstructive pulmonary disease, pulmonary alveolar proteinosis,bleomycin-induced pneumopathy and fibrosis, radiation-induced pulmonaryfibrosis, cystic fibrosis, collagen accumulation in the lungs, and ARDS.GM-CSF inhibitors are useful for treating patients suffering fromvarious skin disorders, including but not limited to dermatitisherpetiformis (Duhring's disease), atopic dermatitis, contactdermatitis, urticaria (including chronic idiopathic urticaria), andautoimmune blistering diseases, including pemphigus vulgaris and bullouspemphigoid. Other diseases treatable with the combination of a GM-CSFinhibitor include myasthenia gravis, sarcoidosis, including pulmonarysarcoidosis, scleroderma, reactive arthritis, hyper IgE syndrome,multiple sclerosis and idiopathic hypereosinophil syndrome. Thetherapeutics of the invention are also useful for treating allergicreactions to medication and as an adjuvant to allergy immunotherapy.

In an embodiment the GM-CSF inhibitor compositions are useful fortreating degenerative conditions of the nervous system, such as multiplesclerosis, relapsing remitting multiple sclerosis, progressive-relapsingmultiple sclerosis, primary and secondary-progressive multiplesclerosis. Targeting GM-CSF is effective in preclinical models ofmultiple sclerosis and therapeutic intervention in the GM-CSF pathway inmultiple sclerosis may reduce CNS inflammation via direct effects onmonocytes, macrophages and dendritic cells, while sparing adaptiveimmunity. GM-CSF knock out mice are resistant to experimental autoimmuneencephalomyelitis (EAE) induction, McQualter, et al., 2001, J. Exp. Med.194:873-881. Adoptive transfer of retrovirally-transduced T cellsexpressing GM-CSF induces exacerbated EAE, Marusic et al., 2002,Neurosci. Lett. 332: 185-9. Adoptive transfer of GM-CSF knock out Tcells fails to induce EAE, Ponomarev et al., 2007, J. Immunol.,178:39-48. Applicants have shown that prophylactic treatment withanti-murine GM-CSF antibody in the SJL-PLP₁₃₉₋₁₅₁EAE model ofrelapsing-remitting multiple sclerosis significantly delayed onset andreduced incidence of disease and reduced both weight loss and meanclinical score, compared to treatment with an isotype control monoclonalantibody, see FIGS. 1 and 2. Therapeutic treatment of SJL/PLP 125-151EAE with anti-mGM-CSF monoclonal antibody significantly reduced diseaseseverity and CNS inflammation and accelerated recovery. Prophylactic andtherapeutic treatment with anti-mGM-CSF monoclonal antibody inSJL-PLP₁₃₉₋₁₅₁ AT-EAE reduced mean clinical score compared to treatmentwith an isotype control monoclonal antibody, see FIG. 2. GM-CSFinhibitor compositions can be used alone or in combination with otherdrugs, for example interferon β-1a (AVONEX®; Biogen-Idec and REBIF® EDMSerono, Inc., Pfizer, Inc.), interferon β-1b (BETASERON®; Bayer HealthCare.), glatiramer acetate (COPAXONE®; Teva Pharmaceuticals) and/oranti-VLA4 mAb (TYSABRI®, Biogen-Idec, Elan).

In one embodiment of the invention, the various medical disordersdisclosed herein as being treatable with GM-CSF inhibitors (e.g., GM-CSFantibodies) are treated in combination with another cytokine or cytokineinhibitor. For example, a GM-CSF inhibitor may be administered in acomposition that also contains a compound that inhibits the interactionof other inflammatory cytokines with their receptors. The GM-CSFinhibitor and other cytokine inhibitors may be administered as separatecompositions, and these may be administered by the same or differentroutes. Examples of cytokine inhibitors used in combination with GM-CSFinhibitor include those that antagonize, for example, TGF-beta,IFN-gamma, IL-6 or IL-8 and TNF, particularly TNF-alpha. The combinationof a GM-CSF inhibitor and IL-6 can be used to treat and prevent therecurrence of seizures, including seizures induced by GABA-A receptorantagonism, seizures associated with EEG ictal episodes and motor limbicseizures occurring during status epilepticus. Further, the combinationof GM-CSF inhibitor and IFN-gamma-1b and/or a c-Kit inhibitor is usefulin treating idiopathic pulmonary fibrosis and cystic fibrosis. Othercombinations for treating diseases include the use of GM-CSF inhibitorwith compounds that interfere with the binding of RANK and RANK-ligand,such as RANK-ligand inhibitors, or soluble forms of RANK, includingRANK:Fc. For example, the combination of GM-CSF inhibitor and RANK:Fcare useful for preventing bone destruction in various settings includingbut not limited to various rheumatic disorders, osteoporosis, multiplemyeloma or other malignancies that cause bone degeneration, oranti-tumor therapy aimed at preventing metastasis to bone, or bonedestruction associated with prosthesis wear debris or withperiodontitis.

The disclosed GM-CSF inhibitors, compositions and combination therapiesdescribed herein are useful in medicines for treating side effectsand/or complications resulting from bacterial, viral or protozoalinfections. According to this embodiment, when an infection triggers anover stimulation of the immune system such that production and/oractivity of GM-CSF results in negative effects on the patient, treatmentwith a GM-CSF inhibitor in patients subject to an infection is useful toameliorate these side effects and/or complications associated with theinfection or therapeutics used to treat the infection. Non limitingexamples of such infectious agents and infections are Mycoplasmapneumonia, AIDS and conditions associated with AIDS and/or related toAIDS, such as AIDS dementia complex, AIDS associated wasting,lipidistrophy due to antiretroviral therapy; CMV (cytomegalovirus),Kaposi's sarcoma; protozoal diseases, including malaria andschistosomiasis; erythema nodosum leprosum; bacterial or viralmeningitis; tuberculosis, including pulmonary tuberculosis; andpneumonitis secondary to a bacterial or viral infection; louse-bornerelapsing fevers, such as that caused by Borrelia recurrentis; Herpesviruses, such as herpetic stromal keratitis, corneal lesions; andvirus-induced corneal disorders; human papillomavirus infections;influenza infection and infectious mononucleosis.

Cardiovascular disorders and injuries are treatable and/or preventablewith the disclosed GM-CSF inhibitors, pharmaceutical compositions orcombination therapies. In particularly cardiovascular disorders aretreatable with GM-CSF inhibitor compositions, alone or in combinationwith TNF inhibitors (e.g. ENBREL) and or other agents as describedabove. Cardiovascular disorders thus treatable include aortic aneurysms;including abdominal aortic aneurysms, acute coronary syndrome,arteritis; vascular occlusion, including cerebral artery occlusion;complications of coronary by-pass surgery; ischemia/reperfusion injury;heart disease, including atherosclerotic heart disease, myocarditis,including chronic autoimmune myocarditis and viral myocarditis; heartfailure, including chronic heart failure, congestive heart failure,cachexia of heart failure; myocardial infarction; restenosis and/oratherosclerosis after heart surgery or after carotid artery balloonangioplastic procedures; silent myocardial ischemia; left ventricularpump dysfunction, post implantation complications of left ventricularassist devices; Raynaud's phenomena; thrombophlebitis; vasculitis,including Kawasaki's vasculitis; veno-occlusive disease, giant cellarteritis, Wegener's granulomatosis; mental confusion following cardiopulmonary by pass surgery, and Schoenlein-Henoch purpura. Combinationsof GM-CSF inhibitors, TNF inhibitors and angiogenesis inhibitors (e.g.anti-VEGF) are useful for treating certain cardiovascular diseases suchas aortic aneurysms and tumors.

In addition, the subject GM-CSF inhibitors, compositions and combinationtherapies are used to treat chronic pain conditions, such as chronicpelvic pain, including chronic prostatitis/pelvic pain syndrome. As afurther example, GM-CSF inhibitor and the compositions and combinationtherapies of the invention are used to treat post-herpetic pain.

In addition to human patients, GM-CSF inhibitors are useful in thetreatment of non-human animals, such as pets (dogs, cats, birds,primates, etc.), domestic farm animals (horses cattle, sheep, pigs,birds, etc.), or any animal that suffers from an IL-1-mediatedinflammatory or arthritic condition. In such instances, an appropriatedose may be determined according to the animal's body weight. Forexample, a dose of 0.2-1 mg/kg may be used. Alternatively, the dose isdetermined according to the animal's surface area, an exemplary doseranging from 0.1-20 mg/m2, or more preferably, from 5-12 mg/m2. Forsmall animals, such as dogs or cats, a suitable dose is 0.4 mg/kg.GM-CSF inhibitor (preferably constructed from genes derived from therecipient species), or another soluble IL-1 receptor mimic, isadministered by injection or other suitable route one or more times perweek until the animal's condition is improved, or it may be administeredindefinitely.

2. Diagnostic Methods

The antigen binding proteins of the described can be used for diagnosticpurposes to detect, diagnose, or monitor diseases and/or conditionsassociated with GM-CSF. The disclosed provides for the detection of thepresence of GM-CSF in a sample using classical immunohistologicalmethods known to those of skill in the art (e.g., Tijssen, 1993,Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds R. H. Burdon andP. H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.);Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al.,1987, J. Cell Biol. 105:3087-3096). The detection of GM-CSF can beperformed in vivo or in vitro.

Diagnostic applications provided herein include use of the antigenbinding proteins to detect expression of GM-CSF and binding of theligands to GM-CSF. Examples of methods useful in the detection of thepresence of GM-CSF include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically willbe labeled with a detectable labeling group. Suitable labeling groupsinclude, but are not limited to, the following: radioisotopes orradionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I),fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic groups (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase), chemiluminescent groups, biotinylgroups, or predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags). In someembodiments, the labelling group is coupled to the antigen bindingprotein via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand may be used.

One aspect of the disclosed provides for identifying a cell or cellsthat express GM-CSF. In a specific embodiment, the antigen bindingprotein is labeled with a labeling group and the binding of the labeledantigen binding protein to GM-CSF is detected. In a further specificembodiment, the binding of the antigen binding protein to GM-CSFdetected in vivo. In a further specific embodiment, the GM-CSF antigenbinding protein is isolated and measured using techniques known in theart. See, for example, Harlow and Lane, 1988, Antibodies: A LaboratoryManual, New York: Cold Spring Harbor (ed. 1991 and periodicsupplements); John E. Coligan, ed., 1993, Current Protocols InImmunology New York: John Wiley & Sons.

Another aspect of the disclosed provides for detecting the presence of atest molecule that competes for binding to GM-CSF with the antigenbinding proteins provided. An example of one such assay would involvedetecting the amount of free antigen binding protein in a solutioncontaining an amount of GM-CSF in the presence or absence of the testmolecule. An increase in the amount of free antigen binding protein(i.e., the antigen binding protein not bound to GM-CSF) would indicatethat the test molecule is capable of competing for GM-CSF binding withthe antigen binding protein. In one embodiment, the antigen bindingprotein is labeled with a labeling group. Alternatively, the testmolecule is labeled and the amount of free test molecule is monitored inthe presence and absence of an antigen binding protein.

3. Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Methods of using the antigen binding proteins are also provided. In somemethods, an antigen binding protein is provided to a patient. Theantigen binding protein inhibits binding of GM-CSFR to human GM-CSF. Theadministration of an antigen binding protein in some methods can alsoinhibit autophosphorylation of human GM-CSF by inhibiting binding ofGM-CSFR to human GM-CSF. Further, in certain methods, monocytechemotaxis is reduced by administering an effective amount of at leastone antigen binding protein to a patient. Monocyte migration into tumorsin some methods is inhibited by administering an effective amount of anantigen binding protein. In addition, the accumulation of tumorassociated macrophage in a tumor can be inhibited by administering anantigen binding protein as provided herein.

Pharmaceutical compositions that comprise a therapeutically effectiveamount of one or a plurality of the antigen binding proteins and apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant are also provided. In addition, methods oftreating a patient by administering such pharmaceutical composition areincluded. The term “patient” includes human patients.

Acceptable formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of human GM-CSF antigen binding proteins are provided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counter ions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such asPolysorbate 20, triton, tromethamine, lecithin, cholesterol, tyloxapal);stability enhancing agents (such as sucrose or sorbitol); tonicityenhancing agents (such as alkali metal halides, preferably sodium orpotassium chloride, mannitol sorbitol); delivery vehicles; diluents;excipients and/or pharmaceutical adjuvants. See, Remington'sPharmaceutical Sciences, 18^(th) Edition, (A. R. Genrmo, ed.), 1995,Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins disclosed. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition may be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier maybe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and mayfurther include sorbitol or a suitable substitute therefor. In certainembodiments, Human GM-CSF antigen binding protein compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the human GM-CSFantigen binding protein may be formulated as a lyophilizate usingappropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery.Alternatively, the compositions may be selected for inhalation or fordelivery through the digestive tract, such as orally. Preparation ofsuch pharmaceutically acceptable compositions is within the skill of theart.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions may be provided in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired human GM-CSF antigenbinding protein in a pharmaceutically acceptable vehicle. A particularlysuitable vehicle for parenteral injection is sterile distilled water inwhich the human GM-CSF antigen binding protein is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used; having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantigen binding protein.

Certain pharmaceutical compositions are formulated for inhalation. Insome embodiments, human GM-CSF antigen binding proteins are formulatedas a dry, inhalable powder. In specific embodiments, human GM-CSFantigen binding protein inhalation solutions may also be formulated witha propellant for aerosol delivery. In certain embodiments, solutions maybe nebulized. Pulmonary administration and formulation methods thereforeare further described in PCT Publication No. WO94/20069 and describespulmonary delivery of chemically modified proteins. Some formulationscan be administered orally. Human GM-CSF antigen binding proteins thatare administered in this fashion can be formulated with or withoutcarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule may bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the human GM-CSF antigen binding protein.Diluents, flavorings, low melting point waxes, vegetable oils,lubricants, suspending agents, tablet disintegrating agents, and bindersmay also be employed.

Some pharmaceutical compositions comprise an effective quantity of oneor a plurality of human GM-CSF antigen binding proteins in a mixturewith non-toxic excipients that are suitable for the manufacture oftablets. By dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions may be prepared in unit-dose form.Suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; or binding agents, such as starch, gelatin, oracacia; or lubricating agents such as magnesium stearate, stearic acid,or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving human GM-CSF antigenbinding proteins in sustained- or controlled-delivery formulations.Techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See, for example, PCT Publication No. WO93/15722 that describes controlled release of porous polymericmicroparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481), copolymers of L-glutamicacid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers2:547-556), poly(2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J.Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., 1981, supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for administration can be stored inlyophilized form or in a solution. Parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. Kits for producing asingle-dose administration unit are also provided. Certain kits containa first container having a dried protein and a second container havingan aqueous formulation. In certain embodiments, kits containing singleand multi-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of a human GM-CSF antigen bindingprotein-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the human GM-CSF antigen binding protein isbeing used, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinicians may titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect.

A typical dosage may range from about 0.1 μg/kg to up to about 30 mg/kgor more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 0.1 μg/kg up to about 30 mg/kg,optionally from 1 μg/kg up to about 30 mg/kg, optionally from 10 μg/kgup to about 10 mg/kg, optionally from about 0.1 mg/kg to 5 mg/kg, oroptionally from about 0.3 mg/kg to 3 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular human GM-CSF antigen binding protein in the formulation used.Typically, a clinician administers the composition until a dosage isreached that achieves the desired effect. The composition may thereforebe administered as a single dose, or as two or more doses (which may ormay not contain the same amount of the desired molecule) over time, oras a continuous infusion via an implantation device or catheter.Appropriate dosages may be ascertained through use of appropriatedose-response data. In certain embodiments, the antigen binding proteinscan be administered to patients throughout an extended time period.Chronic administration of an antigen binding protein minimizes theadverse immune or allergic response commonly associated with antigenbinding proteins that are not fully human, for example an antibodyraised against a human antigen in a non-human animal, for example, anon-fully human antibody or non-human antibody produced in a non-humanspecies.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

It also may be desirable to use human GM-CSF antigen binding proteinpharmaceutical compositions according to the disclosed ex vivo. In suchinstances, cells, tissues or organs that have been removed from thepatient are exposed to human GM-CSF antigen binding proteinpharmaceutical compositions after which the cells, tissues and/or organsare subsequently implanted back into the patient.

In particular, human GM-CSF antigen binding proteins can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptide. In certain embodiments, such cells may be animal or humancells, and may be autologous, heterologous, or xenogeneic. In certainembodiments, the cells may be immortalized. In other embodiments, inorder to decrease the chance of an immunological response, the cells maybe encapsulated to avoid infiltration of surrounding tissues. In furtherembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the described. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

EXAMPLES Example 1 Description of GM-CSF and Other Molecules Used forGeneration, Selection and Characterization of Anti-GM-CSF MonoclonalAntibodies

Several different recombinant and native GM-CSF molecules were used togenerate, select and characterize the anti-GM-CSF hybridomas andmonoclonal antibodies.

Human GM-CSF

There are two naturally occurring allelic variants of human GM-CSF whichdiffer by a single amino acid at position 117. Recombinant human GM-CSFmolecules with either threonine (rhGM-CSF-Thr, SEQ ID NO:146) orisoleucine (GM-CSF-Ile, SEQ ID NO:145) at amino acid position 117 wereproduced from transiently transfected CHO or COS cells andaffinity-purified. Native human GM-CSF (nhGM-CSF) was affinity purifiedfrom the supernatant of A431 cells after stimulation with PMA (0.01ug/ml), ionomycin (0.5 ug/ml) and EGF (0.02 ug/ml), from the supernatantof human peripheral blood mononuclear cells (PBMCs) after stimulationwith PMA (10 ng/mL) and ionomycin (500 ng/mL), for 48 hours at 37° C.,or from the supernatant of human small airway epithelial cells (SAEC)stimulated with TNFα (25 ng/ml) and IL-1 (10 ng/ml). E. coli-derivedrhGM-CSF was purchased from R&D Systems (Minneapolis, Minn.).Yeast-derived rhGM-CSF having a substitution of leucine for arginine atamino acid position 23 (Leukine®) was purchased from Berlex, Inc.(Montville, N.J.). E. coli-derived rhGM-CSF-R23L is an E. coli-derivedrhGM-CSF having a substitution of leucine for arginine at amino acidposition 23. Yeast-derived rhGM-CSF nhGM-CSF (Leukine®), E. coli-derivedrhGM-CSF and E. coli-derived rhGM-CSF-R23L demonstrated equivalentGM-CSF activity in the TF-1 STAT5 phosphorylation assay.

Cynomolgus Macaque GM-CSF

Recombinant cynomolgus GM-CSF (rcynoGM-CSF, SEQ ID NO: 53) was producedfrom transfected E. coli. Native cynomolgus GM-CSF (ncynoGM-CSF) wasaffinity purified from the supernatant of minced lung tissue afterstimulation with TNFα (25 ng/ml) and IL-1 (10 ng/ml), or from thesupernatant of cynomolgus PBMCs after stimulation with PMA (10 ng/mL)and ionomycin (500 ng/mL) for 48 hours at 37° C.

Canine GM-CSF

Canine GM-CSF (SEQ ID NO: 54) was produced from transformed E. coli andpurified from inclusion bodies using standard column chromatography

Rabbit GM-CSF

Recombinant rabbit GM-CSF (SEQ ID NO:82) with His tag was produced from2936E cells, and purified by Talon cobalt IMAC by washing with 5 mMimidazole, 20 mM NaPO4, 300 mM NaCl, pH7.2 and eluting with an imidazolegradient (10 mM to 300 mM).

Mouse GM-CSF

Recombinant mouse GM-CSF (SEQ ID NO:58) was generated from transformedyeast cells and purified with three column chromatography steps:SP-Sepharose (capture), C18 (reverse phase purification) andSP-Sepharose (buffer exchange).

Rat GM-CSF

Recombinant rat GM-CSF was purchased from R&D Systems.

Affinity Purification

Affinity purification of GM-CSF from the supernatant of stimulated cellswas performed by cycling supernatant over anti-GMCSF mAb (M8) affinityresin, washing with NaCltrate pH 6.0, and eluting with NaCltrate pH 4.5.The relevant fractions were pooled and buffer exchanged into PBS. Theconcentration of recombinant GM-CSF was determined by OD₂₈₀, while theconcentration of native GM-CSF was determined by ELISA.

Non-GM-CSF Reagents Used for Characterization of Anti-GM-CSF mAb

Recombinant human CSF-1 was purchased from R&D Systems. RecombinanthIL-15 was generated from transfected CHO cells and purified using a twocolumn step purification process. Anion exchange purification wasperformed with Fractogel TMAE 650M loading at 10 mg/ml resin pH 7.5,then washed with 20 mM Hepes pH 7.5, then 20 mM MES pH 6.5, then 100 mMNaCl, 20 mM MES. Recombinant hIL-15 was eluted with 200 mM NaCl, 20 mMMES pH 6.5. Protein was held at 10 mM NaHPO4 pH 2.0 for 1 hour (viralinactivation step) then subjected to a cation exchange purification stepwith Fractogel EMD SO3-650(M) loading at 20 mg/ml resin at 10 mM NaHPO4pH 2.0, washed with 10 mM NaCltrate, 10 mM NaAcetate, 10 mM MES, pH 4.0,and eluted with a gradient elution 10 mM NaCltrate, 10 mM NaAcetate, 10mM MES, pH 6.0 and buffer exchanged into PBS.

Example 2 Generation and Selection of Neutralizing Human Anti-GM-CSFAntibodies

2.1 Immunization and Selection of GM-CSF-Binding Hybridomas

The development of fully human monoclonal antibodies directed againsthuman GM-CSF were obtained using XenoMouse® technology. Two separatecohorts of 10 KL Xenomice each were immunized every 3-4 days for 7 weeks(16 total injections) with either E. coli-derived rhGM-CSF oralternating injections of mammalian cell-derived rhGM-CSF-Ile andrhGM-CSF-Thr. Serum titers were monitored by enzyme-linked immunosorbentassay (ELISA) after the 7^(th) and 11th boosts and spleen cells frommice with the best titers were fused to partner cell lines 4 days afterthe 16^(th) boost in order to generate hybridomas. The resultingpolyclonal hybridoma supernatants were screened by ELISA for binding toGM-CSF and Alpha screen for the presence of human heavy and kappa and/orlambda light chains. The immunization campaign yielded 499 lines withGM-CSF-binding activity that expressed human heavy and light chains.

2.2 Identification of Hybridomas with GM-CSF Neutralizing Activity UsingHGM-CSF-Dependent Cell-Based Bioassays.

499 hybridoma supernatants were further characterized for GM-CSFneutralizing activity using two cell-based bioassays: GM-CSF inducedSTAT5 phosphorylation in TF-1 cells (2.2.1) and GM-CSF-inducedproliferation of AML-5 cells (2.2.2). From these results, 14 hybridomaswere selected for cloning.

2.2.1 Inhibition of GM-CSF-Induced STAT-5 Phosphorylation in TF-1 Cells.

TF-1 cells were propagated at 37° C., 10% CO₂ in IMDM supplemented with5% FBS, 10 mM Hepes, 2 mM L-glutamine, 50 U/mL Penicillin, 50 μg/mLStreptomycin, 55 uM beta-mercaptoethanol, and 10 ng/mL E. coli-derivedrhGM-CSF. One day prior to the assay, TF-1 cells were harvested bycentrifugation at 350×g for 6 minutes and washed 3 times in PBS. Thecells were resuspended at 1×10⁶/mL and cultured overnight in IMDM+0.5%FBS without GM-CSF at 37° C., 10% CO₂. Assays were performed in 2 mL96-well round bottom plates in 100 μL total volume. Hybridomasupernatants were plated in duplicate at 1:4 final dilution andincubated with rhGM-CSF-Ile (0.4 ng/mL final concentration) for 30minutes at 37° C. Serum- and GM-CSF-starved TF-1 cells were harvested,washed in PBS and resuspended in IMDM with 0.5% FBS at 6×10⁶ cells/mL.50 μL of cell suspension was added (3×10⁵ cells/well) and the plateswere incubated for 15 minutes at 37° C. To fix the cells, 25 μL of 10%paraformaldehyde in PBS was added for a final concentration of 2%paraformaldehyde, and the plates were incubated for 15 minutes at 37° C.200 μL IMDM+0.5% FBS was added to the wells to halt fixation, and theplates were centrifuged at 350×g for 7 minutes. The cell supernatant wasremoved and 400 μL of 90% MeOH was slowly added while vigorously mixingthe cells. Following overnight incubation −20° C., the plates were spun,washed with 400 μL PBS/2% FCS and incubated with 50 μL of a 1:5 dilution(in PBS with 2% FBS) of anti-PhosphoSTAT5-Alexa488 (Becton Dickinson612598, Franklin Lakes, N.J.) for 30 minutes at room temperature. Thecells were washed, resuspended in PBS with 2% FBS and transferred toround bottom microtiter plates for flow cytometry analysis using aMultiWell FACScalibur (Becton Dickinson). The percentage of STAT5⁺ cellswas determined using FlowJo FACS analysis software. The percentinhibition of STAT5 phosphorylation by the hybridoma supernatants wascalculated using the following equation:100−({[% STAT5+of A−% STAT5+of B]/[% STAT5+of C−% STAT5+of B]}*100)Where A=cells+hybridoma supernatant+rhGM-CSF, B=cells only,C=cells+rhGM-CSF

Hybridoma supernatants which inhibited GM-CSF-dependent STAT5phosphorylation greater than threshold values determined for each assaywere further characterized for specificity using IL-3-induced STAT5phosphorylation, and for potency against nhGM-CSF and rcynoGM-CSF usingthe TF-1 phosflow bioassay and the AML-5 cell line GM-CSF-inducedproliferation bioassay (2.2.2). A representative experiment showing theGM-CSF-dependent phospho-STAT5 response and the ability of ananti-hGM-CSF antibody (MAB215, R&D Systems) to inhibit STAT5phosphorylation induced by 0.4 ng/mL rhGM-CSF-Ile is shown in FIG. 3.FIG. 4 shows a histogram of percent inhibition of rhGM-CSF-Ile-inducedSTAT5 phosphorylation by hybridoma supernatants from the cohortimmunized with E. coli-derived rhGM-CSF.

2.2.2 Inhibition of GM-CSF-Dependent Proliferation of AML-5 Cells byHybridoma Supernatants.

AML-5 cells were propagated at 37° C., 10% CO₂ in IMDM supplemented with5% FBS, 10 mM Hepes, 2 mM L-glutamine, 50 U/mL Penicillin, 50 μg/mLStreptomycin, 55 μM beta-mercaptoethanol, and 10 ng/mL E. coli-derivedrhGM-CSF. On the day of experiment, AML-5 cells were centrifuged at350×g for 5 min and washed 4 times in PBS to remove residual GM-CSF. Totest for inhibition of GM-CSF- or CSF-1-induced proliferation of AML-5cells, polyclonal hybridoma supernatants were plated in duplicate in96-well flat bottom microtiter plates at 1:10 and/or 1:30 and/or 1:90final dilutions and cytokine was added at previously determined EC90values: A431 cell-derived nhGM-CSF and rcynoGM-CSF at 0.05 ng/mL;rhGM-CSF-Ile and rhGM-CSF-Thr at 0.15 ng/mL; and rhCSF-1 at 10 ng/mL.The antibody/cytokine mixture was incubated for 30 minutes at 37° C.prior to the addition of 50 μL AML-5 cells at 5×10⁴ cells/mL for a totalvolume of 100 μL (2.5×10³ cells/well). The plates were incubated at 37°C., 10% CO₂ for 72 hours.

To detect AML-5 cell proliferation, the plates were pulsed with onemicrocurie of tritiated thymidine, harvested 6 hours later, and read ona liquid scintillation counter. The percent inhibition of AML-5proliferation by hybridoma supernatants was calculated using thefollowing equation:100−({CPM of A/CPM of B}*100)Where A=cells+hybridoma supernatant+cytokine, and B=cells+cytokine

Hybridoma supernatants which potently inhibited both human andcynomolgus GM-CSF-induced, but not human CSF-1-induced, AML-5 cellproliferation at the 1:30 dilution were selected for cloning to generatemonoclonal anti-GM-CSF hybridomas. A representative experiment showingthe GM-CSF-dependent proliferative response and the ability of ananti-hGM-CSF antibody (MAB215, R&D Systems) to inhibit AML-5 cellproliferation induced by 0.15 ng/mL rhGM-CSF-Ile is shown in FIG. 5.

2.3 Generation of Monoclonal Hybridoma Cell Lines

Based on the results from the bioassay screens, 14 polyclonal hybridomalines were selected for cloning to monoclonality by limiting dilution.Cells from the polyclonal hybridoma plates were counted, resuspended at48 cells/mL and diluted to 24 cells/mL, 4.8 cells/mL and 2.4 cells/mL in20 mL media. Cells were plated at 200 μl/well, generating plates of fourdifferent densities for each line cloned (10 cells/well, 5 cells/well, 1cell/well, and 0.5 cells/well). Within two weeks following cloning, theplates were visually inspected under a microscope and supernatants wereharvested from wells from the 1 cell/well and the 0.5 cell/well cloningplates wherein only a single colony was detected. Supernatants werescreened for antigen specificity by ELISA, and antigen positivesupernatants were analyzed for heavy and light chain species and isotypecomposition by ELISA. One to three daughter clones from each line thatdemonstrated single colony growth in the well, antigen specificimmunoreactivity, and human lambda or kappa and IgG combination onlywere kept and frozen. Monoclonality was confirmed by sequence analysisof each daughter clone.

2.4 Characterization of GM-CSF Neutralizing Activity by Anti-GM-CSFAntibodies Purified from Monoclonal Hybridoma Supernatants

2.4.1 Purification of IgG from Hybridoma Supernatants

IgG was affinity purified from monoclonal hybridoma supernatants usingProtein A column chromatography and quantified using a NanoDrop ND-1000UV-Vis Spectrophotometer at A280 (Nanodrop Technologies, Wilmington,Del.).

2.4.2 Inhibition of GM-CSF-Dependent Proliferation of AML-5 Cells byMonoclonal Antibodies.

AML-5 cells were propagated and prepared for the assay as described inExample 2.2.2. To evaluate the ability of monoclonal antibodies purifiedfrom hybridoma supernatants to inhibit GM-CSF- or CSF-1-inducedproliferation of AML-5 cells, antibodies were each titrated in duplicatein 96-well flat bottom plates (8-fold serial dilutions starting at 5μg/mL). Cytokine was added at previously determined EC90 values:nhGM-CSF (A431 or human PBMC-derived) at 0.1 ng/mL; rhGM-CSF-Ile at 0.3ng/mL; rhGM-CSF-Thr at 0.8 ng/mL; rCynoGM-CSF at 0.05 ng/mL; and rhCSF-1at 3 ng/mL. In each experiment, GM-CSF and CSF-1 were also titrated in2-fold serial dilutions to calculate the EC90 value in that experiment.Cytokine and antibody were incubated for 30 minutes at 37° C. prior tothe addition of AML-5 cells at 2.5×10⁴ cells/mL in a total volume of 100μL. The plates were incubated at 37° C., 10% CO₂. After three days, theplates were pulsed with one microcurie of tritiated thymidine, harvested6 hours later, and read on a liquid scintillation counter. The percentinhibition of AML-5 proliferation by mAb purified from hybridomasupernatants was calculated using the following equation:([CPM of A−CPM of B]/[CPM of A−CPM of C])*100Where A=cells+cytokine, B=cells+mAb+cytokine, and C=cells only

Non-linear regression analysis and 50% inhibition of proliferation(IC50) value calculations were generated using Microsoft Excel (Redmond,Wash.). Experiments in which the amount of cytokine used to stimulatecells was within two-fold of its EC90 value were used to calculate theaverage IC50 values of the monoclonal hybridoma antibodies (Table 5).Experiments using one or more clones of the same mAb (as confirmed bysequence) were included in the averages.

TABLE 5 Table of IC50 (nM) values for mAb purified from hybridomasupernatants in the AML-5 proliferation and TF-1 Stat5 phosphorylationassays. Assay Cytokine IgG A IgG B IgG C IgG D IgG E IgG F AML-5rhGM-CSF-Ile 0.302 0.627 0.347 0.176 0.376 0.193 ProliferationrhGM-CSF-Thr 0.286 0.470 0.501 0.315 0.296 0.179 Assay nhGM-CSF (A431)0.414 0.299 0.442 0.299 0.323 0.179 nhGM-CSF (PBMC) 0.235 0.947 0.6591.373 0.575 0.214 rcynoGM-CSF 0.710 0.574 0.496 0.169 0.669 0.897 TF-1Stat5 rhGM-CSF-Ile 0.046 0.060 0.049 0.114 0.042 0.055 PhosphorylationrhGM-CSF-Thr 0.016 0.023 0.027 0.061 0.035 0.027 Assay nhGM-CSF (A431)0.143 0.075 0.074 0.116 0.067 0.081 rcynoGM-CSF 0.057 0.075 0.066 0.0660.274 0.063

As shown in FIG. 6 and Table 5, several monoclonal antibodies fromhybridoma supernatants inhibited GM-CSF-induced, but not CSF-1-induced,AML-5 cell proliferation in a dose-dependent manner. By fitting for thehalf-maximal inhibition of proliferation, monoclonal antibodies had IC₅₀values of <1 nM against the forms of GM-CSF tested in this assay.

2.4.3 Inhibition of GM-CSF-Dependent STAT-5 Phosphorylation in TF-1Cells by Anti-GM-CSF Monoclonal Antibodies Purified from HybridomaSupernatants.

TF-1 cells were propagated and prepared for the assay as described inExample 2.2.1. To evaluate inhibition of GM-CSF- or rhIL-3-induced STAT5phosphorylation of TF-1 cells, monoclonal antibodies purified fromhybridoma supernatants were each titrated in duplicate in 96-wellround-bottom deep well plates (5-fold serial dilutions starting at 2μg/mL). Cytokine was added at previously determined EC90 values:nhGM-CSF (A431) and rcynoGM-CSF at 0.3 ng/mL; rhGM-CSF-Ile andrhGM-CSF-Thr at 0.9 ng/mL; and rhIL-3 at 30 ng/mL. In each assay, GM-CSFand IL-3 were also titrated in 2-fold serial dilutions to calculate theEC90 value in each assay. Cytokine and antibody were incubated for 30minutes at 37° C. prior to the addition of 3×10⁵ cells/mL serum andGM-CSF starved TF-1 cells in a total volume of 100 μL. Cells werestimulated for 15 minutes at 37° C. then fixed, permeabilized andanalyzed for STAT5 phosphorylation as described in Example 2.2.1. Thepercent inhibition of Stat5 phosphorylation by mAb purified from clonalhybridoma supernatant was calculated using the following equation:([% Stat5+of A−% Stat5+of B]/[% Stat5+of A−% Stat5+of C])*100Where A=cells+cytokine, B=cells+mAb+cytokine, and C=cells only

Non-linear regression analysis and half-maximal inhibition ofproliferation (IC50_(hm) values were calculated using GraphPad Prism4.01. Experiments in which the amount of cytokine used to stimulatecells was within two-fold of its EC90 value were used to calculate theaverage IC50 values of the monoclonal antibodies (Table 5).

As shown in FIG. 7 and Table 5, several monoclonal antibodies inhibitedGM-CSF-induced, but not rhIL-3-induced, STAT5 phosphorylation in TF-1cells in a dose-dependent manner. The monoclonal antibodies hadIC50_(hm) values of <0.3 nM against the forms of GM-CSF tested in thisassay.

Example 3 Cloning and Expression of Recombinant Monoclonal Antibodies(mAb) from Transfected Cell Lines

Heavy and light chain variable regions for the antibody clones weresubcloned into a human IgG2 framework and transiently or stablytransfected and expressed in COS (transient transfection) or CHO (stabletransfection) cells. Antibodies expressed by transient transfection inCOS cells were purified from supernatant using 2.2×10 cm MabSelectSurerProtein A binding in TBS, pH 7.4, and elution with 50 mM Citrate, pH3.4+/−0.2. The eluate was adjusted to pH 6.0 using 1M Tris base stock pH8.0 and buffer exchanged into 10 mM Acetate, 9% Sucrose, pH 5.2 usingdialysis. One clone received continued dialysis into a buffer having 10mM KP, 161 mM L-Arg, pH 7.6 and then concentrated to 20 mg/mL.

Antibodies expressed from stable CHO cell line were purified fromsupernatant using 1.1×10 cm MabSelectSure rProtein A binding in TBS, pH7.4 and elution with 100 mM Acetate, pH 3.6. The eluate was bufferexchanged 5 mg into 10 mM Acetate, 9% Sucrose, pH 5.2 using GE desaltingcolumn. 20-30 mg of material was then buffer exchanged into Cellgro PBS,pH 7.2 using GE desalting. Pools were 0.2 micron filtered. The materialfrom a second stable cell transfection was purified a second time andmaintained in the mM Acetate, 9% Sucrose, pH 5.2 buffer.

Example 4 Kinetic Binding Analysis of Recombinant mAb to rhGM-CSF-Ile bySurface Plasmon Resonance

Kinetic binding analysis of anti-GM-CSF recombinant monoclonalantibodies was performed using surface plasmon resonance at 25° C. usinga Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) equipped with aCM4 sensor chip. Goat anti-human IgG capture antibody was covalentlyimmobilized to the chip using standard amine-coupling chemistry withHBS-EP as the running buffer. Briefly, each flow cell was activated for7 minutes with a 1:1 (v/v) mixture of 0.1 M NHS and 0.4 M EDC at a flowrate of 5 μL/min. Goat anti-human IgG at 30 μg/mL in 10 mM sodiumacetate, pH 5.5 was immobilized at a density of ˜3200 RUs on two flowcells. Residual reactive surfaces were deactivated with a 7-minuteinjection of 1 M ethanolamine at 5 μL/min. Fifty μL of 10 mM glycineHCl, pH 1.5 at 100 μL/min was injected 3 times over each flow cell toremove any remaining noncovalently bound capture antibody and tocondition each surface. The running buffer was switched to HBS-EP with0.1 mg/mL BSA and 2 mg/mL CM-Dextran for all remaining steps.

Recombinant anti-GM-CSF mAb at 0.5 μg/mL was injected over one goatanti-human IgG surface for 1.5 minutes at 10 μL/min to obtain a surfacedensity of ˜111 RUs. The remaining goat anti-human IgG surface was leftunmodified as a reference. Five cycles of buffer blanks were initiallyrun to condition the chip surfaces. Recombinant hGM-CSF-Ile samples wereprepared at concentrations of 300, 100, 33.3, 11.1, 3.70, and 1.23 nM intriplicate and injected in random order along with 6 buffer blanks at100 μL/min over both the captured recombinant anti-GM-CSF IgG andreference surfaces. Each complex was allowed to associate for 2.5minutes, and dissociate for 2.5 minutes. In addition, triplicate samplesof 100 nM rhGM-CSF-Ile and buffer blanks were alternately injected overboth surfaces at 100 μL/min and allowed to associate for 2.5 minutes anddissociate for 90 minutes in order to collect more dissociation phasedata. The surfaces were regenerated after each rhGM-CSF-Ile or bufferinjection with a 30-second pulse of 10 mM glycine HCl, pH 1.5 at 100μL/min, followed by a 30-second injection of buffer.

Data was double referenced by subtracting the reference surfaceresponses to remove bulk refractive index changes, and then subtractingthe averaged buffer blank response to remove systematic artifacts fromthe experimental flow cells. Data collected from the 300 nM curves weredeleted from the analysis for lack of kinetic information, as the datalacked curvature and the concentration was ˜6000x the K_(D). The datawas processed and globally fit to a 1:1 interaction model with Scrubber(version 2.0a, BioLogic Software, Campbell, Australia) to obtain kineticrate constants k_(d) and k_(a), and the equilibrium binding constant,K_(D). The results are shown in Table 6.

TABLE 6 Kinetic binding analysis of recombinant mAb to rhGM-CSF-Ile bysurface Plasmon resonance. mAb K_(a) (M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) IgGA 3.34 × 10⁵ 3.03 × 10⁻⁵ 90 IgG B 6.54 × 10⁵ 3.19 × 10⁻⁵ 49 IgB C 8.67 ×10⁵ 7.05 × 10⁻⁵ 81 IgG E 9.12 × 10⁵ 1.16 × 10⁻⁵ 128

Example 5 Cross-Reactivity of Anti-GM-CSF Antibody to GM-CSF from OtherSpecies as Measured by ELISA

The hybridoma mAb clones and a recombinant mAb from above were evaluatedfor ability to bind rhGM-CSF-Ile, rhGM-CSF-Thr, yeast-derived rhGM-CSF(Leukine®), E. coli-derived rhGM-CSF, nhGM-CSF and/or recombinant GM-CSFfrom one or more of the following species: mouse, rat, rabbit, canineand cynomolgus (FIGS. 8A and 8B). Individual wells of a 96-well platewere coated with 50 μL of 1 ug/mL solutions of GM-CSF or control proteinand incubated overnight at 4° C. Plates were washed four times withPBS/Tween then the lead anti-GM-CSF mAb (or control antibodies) wereadded at 2 ug/ml to wells with each of the GM-CSF proteins, incubatedfor 1 hour at room temperature, and washed 4 times with PBS/Tween.HRP-conjugated anti-human IgG at 1:8000 was added, incubated for 1 hourat room temperature, and plates were washed 4 times with PBS/Tween. TMBdeveloper was added, incubated for 10 minutes and plates were read at650 nm on a plate reader. The anti-GM-CSF mAb clones and recombinant mAbbound to rhGM-CSF-Ile, rhGM-CSF-Thr, E. coli-derived rhGM-CSF andrecombinant cynoGM-CSF, but not to mouse, rat or canine GM-CSF. Some,but not all, of the clones also bound to yeast-derived rhGM-CSF(Leukine®) (FIG. 8A). In a separate assay, one anti-GM-CSF mAb clonefrom hybridoma supernatant and the second stable cell line (SCL)transfection bound to PBMC-derived nhGM-CSF, rhGM-CSF-Ile, andrcynoGM-CSF, but not to mouse, rat, rabbit or canine GM-CSF (FIG. 8B).

Example 6 Determination of IC50 Values for Recombinant Anti-GM-CSF mAbin Cell-Based Bioassays and Human Whole Blood Using Multiple GM-CSFMolecules

As described above, fully human recombinant anti-hGM-CSF antibodies wereexpressed from both transiently transfected cells and stable transfectedcell lines. Material was purified from two independent transienttransfections (1^(st) TT and 2^(nd) TT) and two separate harvests of thestable cell lines (1^(st) SCL and 2^(nd) SCL). The following experimentsdescribe the determination of IC50 values for six recombinant antibodiesclones against different forms of human and cynomolgus GM-CSF (Tables7-9).

6.1 Inhibition of GM-CSF-Dependent Proliferation of AML-5 Cells byRecombinant Anti-GM-CSF mAb.

AML-5 proliferation assays were carried out as described in Example2.4.2 using 9-fold serial dilutions of the mAb starting at 10 μg/mL andcytokine at previously determined EC90 concentrations. For theexperiment using the 2^(nd) TT and 1^(st) SCL material (FIG. 9),cytokine was added at the following concentrations: PBMC-derivednhGM-CSF at 0.2 ng/mL; rhGM-CSF-Ile at 0.4 ng/mL; and rCynoGM-CSF at 0.1ng/mL. For other assays, rhGM-CSF-Thr (0.4 ng/mL) and lungtissue-derived ncynoGM-CSF (3 ng/mL) was tested in addition to thecytokines above. In each experiment, the cytokines used were alsotitrated using 2-fold serial dilutions to calculate the EC90 value forthat experiment. Cytokine and antibody were incubated for 30 minutes at37° C. prior to the addition of 2.5×10⁴ AML-5 cells/mL in a total volumeof 100 μL. After 72 hours at 37° C., 10% CO₂, 1 microCurie of tritiatedthymidine was added per well. The cell cultures were harvested 6 hourslater and incorporated tritiated thymidine was measured by liquidscintillation counting. The percent inhibition of AML-5 proliferation byrecombinant mAb was calculated as in Example 2.4.2. Non-linearregression analysis and 50% inhibition of proliferation (IC50) valuecalculations were generated using Microsoft Excel. Experiments in whichthe amount of cytokine used to stimulate cells was within two-fold ofits EC90 value were used to calculate average IC50 values for each ofthe mAb.

All of the transient- and stable cell line-generated recombinantantibodies inhibited GM-CSF-induced AML-5 cell proliferation in adose-dependent manner. All six lead antibodies from transienttransfections inhibited human GM-CSF with IC50 values <0.8 nM andcynomolgus GM-CSF with IC50 values <3.5 nM. All six stable cell linelead antibodies inhibited human GM-CSF with IC50 values <1.5 nM andcynomolgus GM-CSF with IC50 values <3.5 nM. Results for three of theantibodies are shown in FIG. 9. A summary of IC50 values for allantibodies and cytokines tested in the AML-5 proliferation assay areshown in Table 7.

TABLE 7 Table of IC50 (nM) values for recombinant mAb in the AML-5proliferation assay. Anti- Cytokine body n IgG A IgG B IgG C IgG ErhGM-CMS- 1^(st) TT 2 0.545 0.286(n4) 0.258(n4) 0.403 Ile 2^(nd) TT 30.579 0.581 0.380 0.380(n2) 1^(st) SCL 3 0.844 1.192 0.766 0.971(n2)2^(nd) SCL 1 0.966 0.943 0.595 rhGM-CSF- 1^(st) TT 2 0.475 0.540 0.4690.371 Thr 2^(nd) TT 1 0.181 0.200 0.165 0.249 1^(st) SCL 2 0.706 0.5090.557 0.549 nhGM-CSF 1^(st) TT 2 0.264 0.155 0.190 0.247 (PBMC) 2^(nd)TT 2 0.195 0.232 0.312 0.574(n1) 1^(st) SCL 2 0.308 0.270 0.1600.360(n1) 2^(nd) SCL 1 0.251 0.213 0.081 rcynoGM- 1^(st) TT 1 0.3950.072 0.253 0.215 CSF 2^(nd) TT 1 1.607 1.319 0.864 0.693 1^(st) SCL 23.267 1.177 1.624 2.479(n1) 2^(nd) SCL 1 2.488 0.842 1.755 ncynoGM-2^(nd) TT 1 1.530 3.242 1.222 1.331 CSF (lung) 1^(st) SCL 1 1.471 2.6831.696 1.6046.2 Inhibition of GM-CSF-Dependent STAT5 Phosphorylation in TF-1 Cellsby Recombinant Anti-GM-CSF mAb.

TF-1 STAT5 phosphorylation assays were carried out as described inExample 2.4.3 using 6-fold serial dilutions of the mAb starting at 2μg/mL and cytokine at previously determined EC90 concentrations. For theexperiment using the 2^(nd) TT and 2^(nd) SCL material (FIG. 10),cytokine was added at the following concentrations: PBMC-derivednhGM-CSF and rhGM-CSF-Ile at 0.6 ng/mL; rcynoGM-CSF at 0.1 ng/mL. Forthe experiment using the 1^(st) TT material (FIG. 11), supernatant fromstimulated human small airway epithelial cell (SAEC) cultures (finalnhGM-CSF concentrations of 0.2 ng/mL) and supernatant from stimulatedcynomolgus lung cultures (final ncynoGM-CSF concentrations of 0.1 ng/mL)was used. In FIG. 12, supernatant from stimulated cynomolgus PBMCcultures (final GM-CSF concentration of 1 ng/mL) was added to GM-CSF mAbfrom a 2^(nd) SCL. For other assays, rhGM-CSF-Thr at 0.75 ng/mL wastested in addition to the cytokines above. In each experiment, thecytokines used were also titrated in 2-fold serial dilutions tocalculate the EC90 value for that experiment. After 15 minutes, thecells were fixed and permeabilized and the amount of STAT5phosphorylation was detected using an anti-phospho-STAT5 antibody asdescribed above in Examples 2.2.1 and 2.4.3. The percent inhibition ofStat5 phosphorylation by recombinant mAb was calculated as in Example2.4.3. Non-linear regression analysis and half-maximal inhibition ofproliferation (IC50_(hm)) values were calculated using GraphPad Prism4.01. Experiments in which the amount of cytokine used to stimulatecells was within two-fold of its EC90 value were used to calculateaverage IC50 values of the monoclonal antibodies.

6.3 Inhibition of GM-CSF-Dependent Activation of Primary Human Monocytesby Recombinant Anti-GM-CSF mAb.

To evaluate the mAb for their ability to neutralize GM-CSF-inducedmetabolic activity in human monocytes, primary monocytes were isolatedusing a Monocyte Isolation Kit II (Miltenyi Biotech) from leukapheresispacks (Amgen Washington Blood Donor Program). The negatively selectedcells were 90%-95% CD14⁺ cells as assessed by flow cytometry (data notshown). PBMC-derived nhGM-CSF or rhGM-CSF-Ile (0.05 ng/mL) was incubatedfor 30 minutes with 6-fold serial dilutions of the mAb starting at 20μg/mL in 96-well flat bottom plates. CD14⁺ cells (150,000/mL) were addedin 100 μL total media (RPMI supplemented with 10% FCS, 10 mM Hepes, 2 mML-glutamine, 50 U/mL Penicillin, 50 μg/mL Streptomycin, and 55 μMbeta-mercaptoethanol) to the plates and incubated for 5 days at 37° C.,5% CO₂. GM-CSF-induced metabolic activity was assessed by addition of 20μL of a 1:1 mixture of Alamar Blue (BioSource, DAL1025, Invitrogen,Carlsbad, Calif.) and media, and calculating the absorbance at 570-600nm 4-8 hours later. The percent inhibition of human CD14⁺ monocyteactivity by recombinant mAb was calculated using the following equation:([OD₅₇₀₋₆₀₀ of A−OD₅₇₀₋₆₀₀ of B]/[OD₅₇₀₋₆₀₀ of A−OD₅₇₀₋₆₀₀ of C])*100Where A=cells+cytokine, B=cells+mAb+cytokine, and C=cells only

Non-linear regression analysis and 50% inhibition of proliferation(IC50) value calculations were generated using Microsoft Excel.Experiments in which the amount of cytokine used to stimulate cells waswithin two-fold of its EC90 value were used to calculate average IC50values for the mAb (Table 8). The transient- and stable cellline-generated recombinant antibodies inhibited GM-CSF-induced AML-5cell proliferation in a dose-dependent manner. The antibodies inhibitedhuman GM-CSF with IC50_(hm) values <0.13 nM and cynomolgus GM-CSF withIC50_(hm) values <0.31 nM.

TABLE 8 Table of IC50 (nM) values for recombinant mAb in the TF-1 Stat5phosphorylation assay. Anti- Cytokine body n IgG A IgG B IgG C IgG ErhGM-CMS- 1^(st) TT 1 0.117 0.046 0.072 0.101 Ile 2^(nd) TT 2 0.0160.013 0.012 1^(st) SCL 1 0.011 0.008 0.002 2^(nd) SCL 1 0.027 0.0090.011 rhGM-CSF-Thr 1^(st) TT 1 0.114 0.052 0.050 0.068 nhGM-CSF 2^(nd)TT 1 0.009 0.008 0.009 (PBMC) 2^(nd) SCL 1 0.010 0.008 0.009 nhGM-CSF(SAEC 1^(st) TT 1 0.042 0.025 0.022 0.030 supe) rcynoGM-CSF 1^(st) TT 20.050 0.021 0.031 0.052 2^(nd) TT 1 0.230 0.183 0.305 2^(nd) SCL 1 0.1280.149 0.196 ncynoGM-CSF 2^(nd) TT 1 0.179 0.079 0.141 0.191 (lung sup,)ncynoGM-CSF 1^(st) TT 1 0.0022 (PBMC sup.) 2^(nd) SCL 1 0.0004

The transient- and stable cell line-generated recombinant antibodiesinhibited GM-CSF-induced human monocyte activity in a dose-dependentmanner. The lead antibodies, regardless of expression method, inhibitedboth native and recombinant hGM-CSF with IC50 values <0.55 nM. Theresults for the 1^(st) TT material are shown in FIG. 13. A summary ofIC50 values for all antibodies tested in the human monocyte assay areshown in Table 9.

TABLE 9 Table of IC50 (nM) values for recombinant mAb in the humanmonocyte assay. Anti- Cytokine body n IgG A IgG B IgG C IgG E rhGM-CMS-1^(st) TT 1 0.076 0.078 0.034 0.089 Ile 2^(nd) TT 2 0.136 0.096 0.0830.142 1^(st) SCL 1 0.028 0.051 0.046 2^(nd) SCL 1 0.130 0.150 0.089nhGM-CSF 1^(st) TT 1 0.549 0.253 0.301 0.490 (PBMC) 2^(nd) TT 2 0.2100.219 0.161 0.305 1^(st) SCL 2 0.149 0.151 0.085 0.210(n1) 2^(nd) SCL 10.171 0.204 0.1146.4 Inhibition of GM-CSF-Induced ENA-78 or MIP-1 Beta Production inHuman Whole Blood by a Recombinant Anti-GM-CSF Produced from Stable CHOCell Lines

Recombinant hGM-CSF-Ile (final concentration 2 ng/mL) was prepared inRPM′ supplemented with 10% normal human serum, 100 U/mL Penicillin, 100ug/mL Streptomycin, 2 mM L-glutamine and 25 mM Hepes and added to 6-foldserial dilutions of a recombinant mAb from a stable CHO cell linestarting at 100 μg/mL in 96-well flat bottom plates. Human IgG₂isotype-matched monoclonal antibody (anti-KLH) was added to all wellsfor a final total IgG concentration of 100 μg/mL and the plates wereincubated for 30 minutes at 37° C. Cytokine was titrated in 4-foldserial dilutions +/−100 μg/mL isotype-matched human IgG₂ to calculatethe EC90 value for that experiment. Human whole blood was collected intoNa-Heparin Vacutainer tubes (Becton Dickinson) by the Amgen Whole BloodDonor Program and 228 μL (285 μL total volume) blood was added to thewells and mixed with gently pipetting. After a 40 hr incubation at 37°C., 5% CO₂, the plates were spun for 5 minutes at 730×g, and 55 μLplasma was carefully collected and transferred to new 96 well plates andfrozen. Plasma was thawed and duplicate wells were pooled prior toanalysis for ENA78 and MIP-1b by ELISA using the reagents and protocolsfrom hENA78 and hMIP-1b DuoSets from R&D Systems. Non-linear regressionanalysis and half-maximal inhibition of proliferation (IC50_(hm)) valueswere calculated using GraphPad Prism 4.01.

In this human whole blood assay, the recombinant human GM-CSF mAb wasshown to inhibit GM-CSF-induced production of ENA78 and MIP-1b. TheIC50_(hm) value was determined to be 0.155 nM for ENA78 production and0.299 nM for MIP-1b production (FIG. 14).

Example 7 Neutralization of Yeast-Derived rhGM-CSF (Leukine®),A431-Derived nhGM-CSF, E. coli-Derived rhGM-CSF and E. coli-DerivedrhGM-CSF-R23L in the GM-CSF-Induced AML-5 Proliferation Assay HumanMonocyte Bioassay and GM-CSF-Induced TF-1 STAT-5 Phosphorylation in TF-1Cells Assay

The AML-5 proliferation assay was carried out as described in Example2.4.2 using 9-fold serial dilutions of the GM-CSF mAb purified fromhybridoma supernatant starting at 5 μg/mL and cytokine at previouslydetermined EC90 concentrations (E. coli-derived rhGM-CSF at 0.1 ng/mL,yeast-derived rhGM-CSF at 0.05 ng/mL). The cytokines used were alsotitrated using 2-fold serial dilutions to calculate the EC90 value forthat experiment. Cytokine and antibody were incubated for 30 minutes at37° C. prior to the addition of 2.5×10⁴ AML-5 cells/mL in a total volumeof 100 μL. After 72 hours at 37° C., 10% CO₂, 1 microCurie of tritiatedthymidine was added per well. The cell cultures were harvested 6 hourslater and incorporated tritiated thymidine was measured by liquidscintillation counting. As shown in FIG. 15 a, four mAb were able toneutralize the activity of E. coli-derived rhGM-CSF, but notyeast-derived rhGM-CSF (Leukine®).

The human monocyte assay was carried out as described in Example 6.3using 3-fold dilutions of the mAb purified from hybridoma supernatantstarting at 12 μg/mL and cytokine at previously determined EC90concentrations (A431 cell-derived nhGM-CSF and yeast-derived rhGM-CSF at0.04 ng/mL). The cytokines used were also titrated using 2-fold serialdilutions to calculate the EC90 value for that experiment. Cytokine andantibody were incubated together for 30 minutes at 37° C. prior to theaddition of 1.5×10⁵ human CD14⁺ cells/mL in a total volume of 100 μL.The plates were incubated for 5 days at 37° C. in 5% CO₂ andGM-CSF-induced metabolic activity was assessed by measuring thereduction of Alamar Blue. For both assays, non-linear regressionanalysis and 50% inhibition of proliferation (IC50) value calculationswere generated using Microsoft Excel. Experiments in which the amount ofcytokine used to stimulate cells was within two-fold of its EC90 valuewere included in analysis. As shown in FIG. 15 b, four mAb were able toneutralize the activity of A431 cell-derived rhGM-CSF, but notyeast-derived rhGM-CSF (Leukine®).

The TF-1 STAT5 phosphorylation assay was carried out as described inExample 2.2.1 using recombinant anti-GM-CSF mAb. Into duplicate wells ofa 96 well plate, 6-fold serial dilutions of anti-GM-CSF mAb starting at2 μg/mL and cytokine at previously determined EC₉₀ concentrations (E.coli-derived rhGM-CSF and E. coli-derived rhGM-CSF-R23L at 0.5 ng/mL,yeast-derived rhGM-CSF-R23L (Leukine®) at 0.2 ng/mL). Following 30minute incubation, 3×10⁵ TF-1 cells/ml were added to total volume of 100μL. The plates were incubated for 15 minutes at 37° C. To fix the cells,25 μL of 10% paraformaldehyde in PBS was added for a final concentrationof 2% paraformaldehyde, and the plates were incubated for 10 minutes at37° C. 200 μL IMDM+0.5% FBS, 10 mM Hepes, 2 mM L-glutamine, 50 U/mLPenicillin, 50 μg/mL Streptomycin, 55 uM beta-mercaptoethanol, was addedto the wells to halt fixation, and the plates were centrifuged at 350×gfor 10 minutes. The cell supernatant was removed and 400 μL of 90% MeOHwas slowly added while vigorously mixing the cells. Following overnightincubation at −20° C., the plates were spun, washed with 600 μL PBS/2%FCS and incubated with 50 μL of a 1:5 dilution (in PBS with 2% FBS) ofanti-PhosphoSTAT5-Alexa488 (Becton Dickinson 612598, Franklin Lakes,N.J.) for 30 minutes at room temperature. The cells were washed,resuspended in PBS with 2% FBS and transferred to round bottommicrotiter plates for flow cytometry analysis using a MultiWellFACScalibur (Becton Dickinson). The percentage of STAT5⁺ cells wasdetermined using FlowJo FACS analysis software. The percent inhibitionof STAT5 phosphorylation by the hybridoma supernatants was calculatedusing the following equation:100−{[% STAT5+of A−% STAT5+of B]/[% STAT5+of C−% STAT5+of B]}*100)Where A=cells+hybridoma supernatant+rhGM-CSF, B=cells only,C=cells+rhGM-CSF

In the GM-CSF-induced TF-1 cell pSTAT5 assay, E. coli-derivedrhGM-CSF-R23L exhibited an EC90 (0.384 ng/mL) comparable to that ofyeast-derived rhGM-CSF-R23L (Leukine®) (0.130 ng/mL) and E. coli-derivedrhGM-CSF (0.298 ng/mL). As shown in FIG. 16, anti-GM-CSF mAb neutralizedE. coli-derived rhGM-CSF but not yeast-derived (Leukine®) orunglycosylated E. coli-derived rhGM-CSF-R23L. This suggests that thebasis for this distinction is due to the same single amino aciddifference (leucine to arginine at position 23) in the primary sequenceof the yeast-derived rhGM-CSF-R23L (Leukine®) and E. coli rhGM-CSF-R23L,compared to native GM-CSF, rather than glycosylation difference due toyeast expression.

Example 8 Epitope Binning of Lead Six Anti-GM-CSF Monoclonal Antibodiesby Binding Competition

Epitope binning was performed using a binding competition assay in whichone labeled mAb competed with excess amounts of other unlabeled mAb forbinding to rhGM-CSF-Ile. Antibodies which competed with one another wereassigned to the same bin. Five of the six lead anti-GM-CSF mAb competedwith each other for binding to rhGM-CSF, while one did not.

What is claimed is:
 1. A nucleic acid molecule encoding an isolatedantigen binding protein that binds granulocyte macrophage colonystimulating factor (GM-CSF) comprising: A) a CDRH1 of SEQ ID NO:22, aCDRH2 of SEQ ID NO: 23 and a CDRH3 of SEQ ID NO:24, and B) a CDRL1 ofSEQ ID NO:16, a CDRL2 of SEQ ID NO:17, and a CDRL3 of SEQ ID NO:
 18. 2.The nucleic acid molecule according to claim 1, wherein said nucleicacid molecule is operably linked to a control sequence.
 3. A vectorcomprising a nucleic acid molecule according to claim
 1. 4. A vectorcomprising a nucleic acid molecule according to claim
 2. 5. A host cellcomprising the nucleic acid molecule according to claim
 2. 6. A hostcell comprising the vector according to claim
 3. 7. A method of makingthe antigen binding protein according to claim 1, comprising the step ofpreparing said antigen binding protein from a host cell that secretessaid antigen binding protein.